Efficacy of T cell assays for the diagnosis of primary defects in cytotoxic lymphocyte exocytosis.

Primary hemophagocytic lymphohistiocytosis (HLH) is a life-threatening disorder associated with autosomal recessive variants in genes required for perforin-mediated lymphocyte cytotoxicity. A rapid diagnosis is crucial for successful treatment. Although defective cytotoxic T lymphocyte (CTL) function causes pathogenesis, quantification of natural killer (NK) cell exocytosis triggered by K562 target cells currently represents a standard diagnostic procedure for primary HLH. We have prospectively evaluated different lymphocyte exocytosis assays in 213 patients referred for evaluation for suspected HLH and related hyperinflammatory syndromes. A total of 138 patients received a molecular diagnosis consistent with primary HLH. Compared to routine K562 cell-based assays, assessment of Fc receptor-triggered NK-cell and T cell receptor-triggered CTL exocytosis displayed higher sensitivity and improved specificity for the diagnosis of primary HLH, with these assays combined providing a sensitivity of 100% and specificity of 98.3%. By comparison, NK-cell exocytosis following K562 target cell stimulation displayed a higher inter-individual variability, in part explained by differences in NK-cell differentiation or large functional reductions following shipment. We thus recommend combined analysis of T cell receptor-triggered CTL and Fc receptor-triggered NK-cell exocytosis for the diagnosis of patients with suspected familial HLH or atypical manifestations of congenital defects in lymphocyte exocytosis.

Identification of the molecular etiology in rare congenital hemolytic anemias using next-generation sequencing with exome-based copy number variant analysis.

OBJECTIVES: In congenital hemolytic anemias (CHA), it is not always possible to determine the specific diagnosis by evaluating clinical findings and conventional laboratory tests. The aim of this study is to evaluate the utility of next-generation sequencing (NGS) and clinical-exome-based copy number variant (CNV) analysis in patients with CHA. METHODS: One hundred and forty-three CHA cases from 115 unrelated families referred for molecular analysis were enrolled in the study. Molecular analysis was performed using two different clinical exome panels in 130 patients, and whole-exome sequencing in nine patients. Exome-based CNV calling was incorporated into the traditional single-nucleotide variant and small insertion/deletion analysis pipeline for NGS data in 92 cases. In four patients from the same family, the PK Gypsy variant was investigated using long-range polymerase chain reaction. RESULTS: Molecular diagnosis was established in 86% of the study group. The most frequently mutated genes were SPTB (31.7%) and PKLR (28.5%). CNV analysis of 92 cases revealed that three patients had different sizes of large deletions in the SPTB and six patients had a deletion in the PKLR. CONCLUSIONS: In this study, NGS provided a high molecular diagnostic rate in cases with rare CHA. Analysis of the CNVs contributed to the diagnostic success.

Changing Disease Course of Crimean-Congo Hemorrhagic Fever in Children, Turkey.

Crimean-Congo hemorrhagic fever (CCHF), endemic in certain regions of the world, is listed as a priority disease with pandemic potential. Since CCHF was first identified in Turkey, children have been known to experience milder disease than adults. However, during the COVID-19 pandemic, we observed an unusually severe disease course, including hemophagocytic lymphohistiocytosis (HLH). We examined cytokine/chemokine profiles of 9/12 case-patients compared with healthy controls at 3 time intervals. Interferon pathway-related cytokines/chemokines, including interleukin (IL) 18, macrophage inflammatory protein 3α, and IL-33, were elevated, but tumor necrosis factor-α, IL-6, CXCL8 (formerly IL-8), and cytokines acting through C-C chemokine receptor 2 and CCR5 were lower among case-patients than controls. Interferon pathway activation and cytokines/chemokines acting through CCR2 and CCR5 improved health results among children with severe CCHF. Children can experience severe CCHF, including HLH, and HLH secondary to CCHF can be successfully treated with intravenous immunoglobulin and steroid therapy.

Intestinal mycobiota composition and changes in children with thalassemia who underwent allogeneic hematopoietic stem cell transplantation.

BACKGROUND: Allogeneic hematopoietic stem cell transplantation (HSCT) alters the diversity of the intestinal bacterial microbiota. This study aimed to evaluate human mycobiota composition pre-HSCT and post-HSCT in children with thalassemia. METHOD: Ten children with thalassemia undergoing allogeneic HSCT were enrolled. The stool samples were collected before the transplantation regimen, before the transplant day, and +15, +30 days, and three months after transplantation. Stool samples were also collected from the donor and the patient's caregivers. Gut mycobiota composition was evaluated with metagenomic analysis. RESULTS: Pretransplant mycobiota of children with thalassemia (the predominant genus was Saccharomyces, 64.1%) has been shown to approximate the diverse mycobiota compositions of healthy adult donors but becomes altered (lower diversity) following transplant procedures. Three months after HSCT, phyla Ascomycota and Basidiomycota were 83.4% and 15.6%, respectively. The predominant species were Saccaharomyces_uc and Saccharomyces cerevisiae (phylum Ascomycota); we also observed Malassezia restricta and Malassezia globosa (phylum Basidiomycota) (∼13%). On day 90 after HSCT, we observed 65.3% M. restricta and 18.4% M. globosa predominance at the species level in a four-year-old boy with acute graft-versus-host disease (GVHD) (skin and gut involvement) 19 days after transplantation included. CONCLUSION: The mycobiota composition of children with thalassemia altered after HSCT. We observed Malassezia predominance in a child with GVHD. Further studies in children with GVHD will identify this situation.

Liposomal amphotericin B related late-onset hyperphosphatemia in a pediatric patient with acute myeloid leukemia.

INTRODUCTION: Children with acute myeloid leukemia (AML) are at risk for serious electrolyte abnormalities. CASE REPORT: We reported a case of hyperphosphatemia in a child with acute myeloid leukemia who received liposomal amphotericin B (AMBL) for the treatment of an invasive fungal infection. The findings of this case suggest that cumulative dose accumulation due to long term AMBL treatment may result in late-onset hyperphosphatemia. MANAGEMENT AND OUTCOME: This is the first case report in the literature that of late-onset hyperphosphatemia (day 56) in a patient with low-dose AMBL treatment (3-5 mg/kg/day) and normal renal function. DISCUSSION: We highlight the importance of increasing awareness of AMBL related hyperphosphatemia among healthcare providers.

Prognostic factors for survival in children who relapsed after allogeneic hematopoietic stem cell transplantation for acute leukemia.

BACKGROUND: Post-transplant relapse has a dismal prognosis in children with acute leukemia undergoing allogeneic hematopoietic stem cell transplantation (allo-HSCT). Data on risk factors, treatment options, and outcomes are limited. PROCEDURE: In this retrospective multicenter study in which a questionnaire was sent to all pediatric transplant centers reporting relapse after allo-HSCT for a cohort of 938 children with acute leukemia, we analyzed 255 children with relapse of acute leukemia after their first allo-HSCT. RESULTS: The median interval from transplantation to relapse was 180 days, and the median follow-up from relapse to the last follow-up was 1844 days. The 3-year overall survival (OS) rate was 12.0%. The main cause of death was disease progression or subsequent relapse (82.6%). The majority of children received salvage treatment with curative intent without a second HSCT (67.8%), 22.0% of children underwent a second allo-HSCT, and 10.2% received palliative therapy. Isolated extramedullary relapse (hazard ratio (HR): 0.607, P = .011) and relapse earlier than 365 days post-transplantation (HR: 2.101, P < .001 for 0-180 days; HR: 1.522, P = .041 for 181-365 days) were found in multivariate analysis to be significant prognostic factors for outcome. The type of salvage therapy in chemosensitive relapse was identified as a significant prognostic factor for OS. CONCLUSION: A salvage approach with curative intent may be considered for patients with post-transplant relapse, even if they relapse in the first year post-transplantation. For sustainable remission, a second allo-HSCT may be recommended for patients who achieve complete remission after reinduction treatment.

Genome instability is a consequence of transcription deficiency in patients with bone marrow failure harboring biallelic ERCC6L2 variants.

Biallelic variants in the ERCC excision repair 6 like 2 gene (ERCC6L2) are known to cause bone marrow failure (BMF) due to defects in DNA repair and mitochondrial function. Here, we report on eight cases of BMF from five families harboring biallelic variants in ERCC6L2, two of whom present with myelodysplasia. We confirm that ERCC6L2 patients' lymphoblastoid cell lines (LCLs) are hypersensitive to DNA-damaging agents that specifically activate the transcription coupled nucleotide excision repair (TCNER) pathway. Interestingly, patients' LCLs are also hypersensitive to transcription inhibitors that interfere with RNA polymerase II (RNA Pol II) and display an abnormal delay in transcription recovery. Using affinity-based mass spectrometry we found that ERCC6L2 interacts with DNA-dependent protein kinase (DNA-PK), a regulatory component of the RNA Pol II transcription complex. Chromatin immunoprecipitation PCR studies revealed ERCC6L2 occupancy on gene bodies along with RNA Pol II and DNA-PK. Patients' LCLs fail to terminate transcript elongation accurately upon DNA damage and display a significant increase in nuclear DNA-RNA hybrids (R loops). Collectively, we conclude that ERCC6L2 is involved in regulating RNA Pol II-mediated transcription via its interaction with DNA-PK to resolve R loops and minimize transcription-associated genome instability. The inherited BMF syndrome caused by biallelic variants in ERCC6L2 can be considered as a primary transcription deficiency rather than a DNA repair defect.

Central nervous system lesions in Fanconi anemia: Experience from a research center for Fanconi anemia patients.

BACKGROUND: Brain atrophy, abnormal pituitary morphology, corpus callosum, and posterior fossa abnormalities have been described in patients with Fanconi anemia (FA). We aimed to provide an overview of cranial neuroimaging findings and to evaluate the clinical implications in FA patients. PROCEDURE: Cranial magnetic resonance imaging (MRI) studies of 34 patients with FA were retrospectively evaluated, and patients' clinical data were correlated with the imaging findings. RESULTS: The patients' median age was 17.6 (range, 3.9-28) years. At least one pathological brain imaging finding was demonstrated in 22 (65%) patients. These findings included corpus callosum abnormalities and other related supratentorial malformations in nine, pituitary abnormalities in eight, craniovertebral junction and posterior fossa abnormalities in eight, vascular lesions in six, and intracerebral calcifications in two patients. Among the 22 patients who had abnormal cranial MRI findings, six (27%) had mild to moderate intellectual disability (ID), three (14%) had epilepsy, one (5%) had mild hearing loss, and one patient (5%) had hemiplegia. Among these 34 patients, 14 (41%) were transfusion dependent. There was no significant difference between patients with congenital and acquired neuroimaging findings and patients with normal neuroimaging regarding transfusion dependency. CONCLUSIONS: Acquired abnormalities in brain tissue, such as white matter intensity changes, white matter T2 hyperintense discrete foci, or infarcts along with congenital abnormalities, were identified in this study. Variable abnormal brain imaging findings in FA patients, although some were not associated with clinical neurological manifestations, suggest that brain imaging could be part of screening in FA.

Hb H Disease Diagnosed During Adolescent Pregnancy.

Hb H disease is a moderate to severe form of α-thalassemia (α-thal). Patients with Hb H disease may become symptomatic, especially during infections and pregnancy, and may require transfusions. Herein, we present a 16-year-old female with Hb H disease who was initially diagnosed during adolescent pregnancy and was found to carry the -α3.7/-(α)20.5 deletions. The relatively mild presentation of this case highlights the milder phenotypic consequences of deletional α mutations. The case describes the screening and management of pregnancy with Hb H disease. Additionally, this case demonstrates that screening of some undiagnosed inherited blood disorders is important during pregnancy.

A Case With Pyruvate Kinase Deficiency Remarkably Sensitive to Heat.

Pyruvate kinase (PK) deficiency is the most common defect of the glycolytic pathway leading to congenital hemolytic anemia. We present the case of an 18-year-old boy with chronic nonspherocytic hemolytic anemia, who had remarkable sensitivity to heat. Moreover, the patient showed clinical impairment in the last year. For this reason, we excluded the immunologic or infectious nature (malaria, babesia), which may play a role in the worsening of anemia. Red blood cell enzyme assay showed the presence of a significant increase in other enzyme activities, except for PK, suggesting a PK deficiency in the patient. The molecular analysis of the PK-LR gene revealed the presence of a novel homozygote missense mutation (c.581G>C, p.Arg194Pro). The mutant enzyme displayed heat instability. In addition, we analyzed bilirubin uridine diphosphate (UDP)-glucuronosyltransferase 1A1 gene that revealed a heterozygous state ([TA]6/[TA]7). After a clear diagnosis of PK deficiency, the patient underwent splenectomy.

Two versus three day upfront use of granulocyte-colony stimulating factor in healthy bone marrow donors for pediatric bone marrow transplantation.

In order to decrease donors' exposure to granulocyte-colony stimulating factor (G-CSF), we compared the effect of two versus three days of G-CSF priming on CD34+ yield in bone marrow (BM) harvest. Although the number of BM-CD34+ cells was higher in 3day G-CSF priming, we achieved the same number of CD34+ cells per recipient's weight in 2day G-CSF priming group, too. In addition, the number of total nucleated cells (TNC) harvested from BM were similar with two or three day regimen. But mononuclear cells (MNC) of the BM graft was higher in the 3day G-CSF priming group. Similar to CD34+ cell numbers, BM harvest yielded similar TNC, and MNC numbers per kilogram of the recipient. We also found that, young donors (≤10year) had more peripheral blood MNC, bone marrow MNC and CD34+ cell numbers. Another interesting finding of this study was obtaining adequate number of peripheral blood stem cells for leukapheresis with three day G-CSF administration. Since engrafment times were also similar in two groups, we concluded that 2-days G-CSF priming was resulted in sufficient mobilization of BM stem cells.

Secondary antifungal prophylaxis in pediatric hematopoietic stem cell transplants.

Invasive fungal infections (IFIs) constitute a leading cause of morbidity and infection-related mortality among hematopoietic stem cell transplant (HSCT) recipients. With the use of secondary prophylaxis, a history of IFI is not an absolute contraindication to allo-HSCT. However, still, IFI recurrence remains a risk factor for transplant-related mortality. In this study, of the 105 children undergoing HSCT between April 2010 and February 2013, 10 patients who had IFI history before transplantation and had undergone allo-HSCT were evaluated retrospectively to investigate results of secondary prophylaxis. In conclusion, our study shows that amphotericin B and caspofungin was successful as secondary antifungal prophylaxis agents with no relapse of IFI. In addition, after engraftment, secondary prophylaxis was continued with voriconazole orally in 4 patients that yielded good results.

Massive splenic infarction and portal vein thrombosis in children with chronic myeloid leukemia.

Massive splenic infarction and portal vein thrombosis (PVT) due to chronic myeloid leukemia (CML) is extremely rare. We describe 2 children who were presented with massive splenic infarction and PVT in the course of CML. Massive splenic infarction and PVT treated with splenectomy in one and with medical treatment in another in whom PVT resolved by cytoreductive treatment, led to downsizing of spleen or splenectomy. Splenic infarct and PVT should be considered in CML patients with long-lasting severe abdominal pain despite appropriate medical attempts. Splenectomy should be spared for persistent symptoms and complications.

Use of plerixafor for peripheral blood stem cell mobilization failure in children.

BACKGROUND: Peripheral blood stem cell mobilization is usually performed following chemotherapy plus G-CSF in children. This standard approach may not be successful in some heavily pretreated patients undergoing mobilization. Plerixafor (AMD3100) has been used in adults as a second line mobilizing agent. Our aim is to analyze our experiences with plerixafor in children. METHODS: We retrospectively evaluated three children who received plerixafor as a second line stem cell mobilizing agent in our department in the 2010-2012 period. Data including age, sex, diagnosis, previous chemotherapy, radiotherapy details, previous harvest attempts, adverse reaction, and harvest outcome were analyzed. RESULTS: We used plerixafor in combination with G-CSF and chemotherapy or with only G-CSF seven times in three patients. All three patients were treated with different multiple chemotherapy regimens prior to stem cell harvest and failed earlier mobilization with chemotherapy plus G-CSF. The diagnoses were relapsed Hodgkin lymphoma in two and recurrent Ewing's sarcoma in one patient. We used plerixafor in combination with G-CSF and chemotherapy or with only G-CSF seven times in three patients. The harvest was successful in four of seven attempts. No adverse reaction was observed in the patients. CONCLUSION: The success rate is four out of seven attempts (57%) in our group. Although the data regarding the use of plerixafor in children is scarce, our experience also supports its use in poor mobilizer children. The use of plerixafor in children results in effective increases in peripheral stem cell counts and reduces the risk of mobilization failure.

Homozygosity for HBA1: c.179G > A: Hb Adana in an infant.

Hb Adana (HBA1: c.179G > A) is a very rare, unstable form of α-globin variant that results from deficient synthesis of functional α chains. We present a 2-month-old boy with hypochromic microcytic anemia, and remarkable anisocytosis, target cells and basophilic stippling on his peripheral blood smear. α-Globin gene analysis of the patient determined homozygosity for HBA1: c.179G > A, a mutation known as Hb Adana. On his follow-up visit, hemoglobin (Hb) levels were stable at 9.0-9.5 g/dL and mean corpuscular volume (MCV) was 62.2-62.5 fL without the need for a blood transfusion. Clinical and hematological findings of our case were comparable to Hb H (ß4) or ß-thalassemia intermedia (ß-TI)-like phenotypes, despite the fact that he carried an α1 gene mutation. Heterozygosity for the HBA1: c.179G > A mutation may also lead to microcytosis only as seen in his parents. According to our current knowledge, this is the first described case with homozygosity for the Hb Adana mutation, carried on the α1 gene. The relatively mild presentation of the case highlights the milder phenotypic consequences of nondeletional α mutations in the α1 vs. the α2 gene.

Iron Deficiency Anemia in Infancy, Childhood, and Adolescence.

Iron deficiency is the most common nutrient deficiency in the world. Epidemiological data indi- cate that iron deficiency is more prevalent in infants, in preschool-children, and in adolescents. Etiologies of iron deficiency in children include inadequate dietary iron intake, which is the most common cause, as well as increased iron requirements, bleeding, and intestinal iron absorption disorders. Iron deficiency can lead to symptoms related to anemia, may interfere with neuro- development and may cause skin, hair, nail, and gastrointestinal problems. This review focuses on the causes, signs, symptoms, diagnosis and management of iron deficiency in infancy, child- hood, and adolescence.

A rare case of Klippel-Trenaunay syndrome presenting with chronic myeloid leukemia.

BACKGROUND: Klippel-Trenaunay syndrome (KTS) is an overgrowth syndrome associated with capillary/venous/ lymphatic malformations with limb hypertrophy and cancer risk. Various cancers, mostly Wilms tumor, have been reported in patients with KTS, but not leukemia. Chronic myeloid leukemia (CML) is also a rare disease in children, where there is no known disease or syndrome to predispose to CML. CASE: We report a case of CML incidentally diagnosed in a child with KTS when he was bleeding from surgery of the left groin for vascular malformation. CONCLUSIONS: This case reflects the variety of cancer types that may accompany KTS and provides information about CML prognosis in such patients.