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.

Central nervous system thrombosis in pediatric acute lymphoblastic leukemia in Turkey: A multicenter study.

BACKGROUND: In patients with acute lymphoblastic leukemia (ALL), the risk of thromboembolism increases due to hemostatic changes secondary to the primary disease and due to treatment-related factors. In this multicenter study, we aimed to research the frequency of central nervous system (CNS) thrombosis occurring during treatment, hereditary and acquired risk factors, clinical and laboratory features of patients with thrombosis, treatment approaches, and thrombosis-related mortality and morbidity rates in pediatric ALL patients. PROCEDURE: Pediatric patients who developed CNS thrombosis during ALL treatment from 2010 to 2021 were analyzed retrospectively in 25 different Pediatric Hematology Oncology centers in Türkiye. The demographic characteristics of the patients, symptoms associated with thrombosis, the stage of the leukemia treatment during thrombosis, the anticoagulant therapy applied for thrombosis, and the final status of the patients recorded through electronic medical records were determined. RESULTS: Data from 70 patients with CNS thrombosis during treatment, out of 3968 pediatric patients with ALL, were reviewed. The incidence of CNS thrombosis was 1.8% (venous: 1.5 %; arterial: 0.03%). Among patients with CNS thrombosis, 47 had the event in the first 2 months. Low molecular weight heparin (LMWH) was the most commonly used treatment with a median of 6 months (min-max: 3-28 months). No treatment-related complications occurred. Chronic thrombosis findings occurred in four patients (6%). In five (7%) patients who developed cerebral vein thrombosis, neurological sequelae (epilepsy and neurological deficit) remained. One patient died related to thrombosis, and the mortality rate was 1.4%. CONCLUSION: Cerebral venous thrombosis and, less frequently, cerebral arterial thrombosis may develop in patients with ALL. The incidence of CNS thrombosis is higher during induction therapy than during other courses of treatment. Therefore, patients receiving induction therapy should be monitored carefully for clinical findings suggestive of CNS thrombosis.

Impact of TP53 gene variants on prognosis and survival of childhood acute lymphoblastic leukemia.

The tumor suppressor protein 53 (TP53) gene is one of the most studied genes in cancer. Although TP53 variants are rare events in acute leukemia, recent observations showed that relapse samples might harbor TP53 variants. Here, we aimed to determine TP53 variants (hotspot region, exon 4-11) in childhood acute lymphoblastic leukemia (B and T-ALL) patients (n = 94) including diagnostic-relapse pairs (n = 15) by amplicon sequencing and evaluate the clinical impact of these variants. In total, nine different (E298*, R283C, R273H, L252F, C229F, I195T, E286G, c.955_956insC, and c.920-1G > C) variants were identified in 17 (18%) childhood ALL patients. c.(920-1G> C) splice site variant and c.(955_956insC) insertion were reported for the first time. In diagnose-relapse pair samples, we identified acquired and/or loss of TP53 variants in the samples at the time of relapse. TP53 variants were found to be more common in T-ALL (37%) than in B-ALL patients (9%). Pathogenic TP53 variants were associated with a shorter overall survival time (p = 0.001).TP53 variants were found to be associated with inferior outcomes in our cohort and can be an independent risk factor for poor prognosis in childhood acute leukemia patients. Identification of low-frequent variants with next-generation sequencing approaches enables better knowledge of the clonal dynamics of ALL.

Pedipacks in the transfusion of pediatric patients to reduce wastage of blood components: an observational study from a tertiary center.

BACKGROUND: Pedipacks prevent wastage of blood components but they are not used efficiently in pediatric clinics. METHODS: Red cell concentrate (RCC) and platelet concentrate volumes transfused in the last eight months in the pediatric clinics were screened. To calculate the wastage of blood components, the number of transfused pedipacks, whole unit RCC, and platelet units were screened from transfusion laboratory digital records to show the number of whole RCC units or platelets units used instead of pedipacks. The study results were shared with physicians and transfusion laboratory staff and they were trained on the subject in meetings. Two years later, the transfusion laboratory records were assessed again to evaluate pedipack usage. A google questionnaire was also submitted to the transfusion laboratories of other hospitals to assess the use of pedipacks. RESULTS: RCC and platelets were used in 82.9% of the transfusions, and 31.2% of RCC and 18.4 % of platelets were transfused to patients ≤12 months. During the study period, 569 pedipacks and 117 random donor or apheresis platelets separated into satellite packs would be required. But only 48 pedipacks of RCCs and 24 units of random donor platelets/apheresis platelets separated into satellite packs were used. After two years, in RCC transfusions of 0-12 month-old patients, the transfusion laboratory release of pedipacks increased to 67.9% from 13.5%. Other centers were not also using pedipacks efficiently. The main reasons were unawareness of the subject, the blood bank delivering two units of pedipacks even when only one unit was ordered and the risk of not using the second pedipack before the expiry date, and the short expiry date of irradiated pedipacks. CONCLUSIONS: By increasing awareness of the subject, the collaboration of the clinic and laboratory and solving bureaucratic problems, rational use of blood components can be achieved.

Zinc finger protein 384 (ZNF384) impact on childhood mixed phenotype acute leukemia and B-cell precursor acute lymphoblastic leukemia.

B-cell precursor acute lymphoblastic leukemia (BCP-ALL) is a heterogeneous malignancy and consists of several genetic abnormalities. Some of these abnormalities are used in clinics for risk calculation and treatment decisions. Patients with ZNF384 rearrangements had a distinct expression profile regardless of their diagnosis, BCP-ALL or mixed phenotype acute leukemia (MPAL) and defined as a new subtype of ALL. In this study, we screened 42 MPAL and 91 BCP-ALL patients for the most common ZNF384 fusions; ZNF384::TCF3, ZNF384::EP300 and ZNF384::TAF15 by using PCR. We identified ZNF384 fusions in 9.5% of MPAL and 7.6% of BCP-ALL. A novel breakpoint was identified in ZNF384::TCF3 fusion in one BCP-ALL patient. T-myeloid MPAL patients showed significantly lower ZNF384 expression compared to lymphoid groups. Patients with ZNF384r had intermediate survival rates based on other subtypes. Prognostic and patient-specific treatment evaluation of ZNF384 fusions in both ALL and MPAL might help to improve risk characterization of patients.

Clinical characteristics of a Turkish family with congenital erythrocytosis due to an EPOR mutation: Is routine phlebotomy indicated in children and adolescents?

Here we present two siblings, a 9-year-old boy and a 15-year-old girl at presentation, with congenital erythrocytosis due to an EPOR c.1316G>A (p.Trp439Term) mutation. The patients had nausea, abdominal pain, and headache when they presented with hemoglobin levels of 23 g/dL and 19.4 g/dL, respectively. Their father, paternal uncle, and probably the paternal aunt and grandmother had congenital erythrocytosis. The siblings generally preferred to visit hospital when hyperviscosity symptoms developed and had intermittent phlebotomies. Their compliance to anti-aggregant and hematinic treatment was not satisfactory. Within the 11-year follow-up period, the siblings had no thrombohemorrhagic complications, whereas their 39-year-old uncle had a stroke. In addition to antiaggregant treatment, phlebotomy during hyperviscosity symptoms may be safe in children and adolescents; routine phlebotomies may be recommended to adults to prevent thrombohemorrhagic complications.

Bleeding characteristics and management of minor surgeries in rare bleeding disorders: report from a Turkish Pediatric Hematology Center.

BACKGROUND AND OBJECTIVES: In this retrospective report the aim was to present the experience about bleeding characteristics and management of minor surgeries in rare bleeding disorders (RBDs). METHODS: Twenty-six patients were included; with Factor (F) V, FV+VIII, VII, FXI deficiency and afibrinogenemia. Six of the patients were asymptomatic. RESULTS: Fifty-three percent of the patients suffered from mucosal bleeding. Life-threatening bleedings were observed only in the patients with afibrinogenemia and good hemostatic control could only be provided with plasma-derived (pd)-fibrinogen concentrate. Twelve of the patients had undergone 17 minor surgeries. In the patients with FVII and FXI deficiencies with plasma F:C activity between 20-47%, there was a history of uneventful tooth extractions, circumcisions and a pilonidal sinus operation performed without any replacement treatment, whereas one patient with plasma F:C activity of FVII 47% had a history of poor hemostatic control during an adeno-tonsillectomy operation. Although some of these patients were asymptomatic to be on the safe side, minor operations were performed with preoperative administration of one dose of (pd)-fibrinogen concentrate to one afibrinogenemia patient, recombinant active FVII (rFVIIa) to 2 FVII deficient patients and fresh frozen plasma (FFP) to 3 FXI deficient and 1 FVII deficient patients plus postoperatively tranexamic acid (TXA) for 5-7 days. Only with one dose of the replacement therapy just before surgeries good hemostatic control was achieved and none of them had bleeding neither during nor after the surgeries. CONCLUSION: We suggest that minor operations must be performed with preoperative replacement therapies plus 5-7 days of antifibrinolytics under close observation of the hematologist and the surgeon.

Nonsyndromic Juvenile Myelomonocytic Leukemia With PTPN11 Mutation in a 9-Year-old Girl.

A 9.5-year-old girl with malaise, fever, massive hepatosplenomegaly, anemia, leukocytosis (37.9 × 10(9)/L), monocytosis (1.48 × 10(9)/L), and thrombocytopenia is presented. Hemoglobin F was increased (18%). Bone marrow erythroid/myeloid ratio was 40/1 with 7% myeloblast and 5% monocyte suggesting erythroleukemia or juvenile myelomonocytic leukemia (JMML). The patient had a fulminant course with respiratory compromise and died in 2 weeks before heterozygous somatic mutation in the PTPN11 gene was shown. JMML must be considered also in the patients older than 6 years. A cytopenic phase may precede JMML. Leucocytosis may be transient and there may be predominance of erythroid precursors in the bone marrow.

The Efficacy and Safety of Procedural Sedoanalgesia with Midazolam and Ketamine in Pediatric Hematology.

OBJECTIVE: The aim of this study is to investigate the efficacy and safety of sedoanalgesia performed outside the operating room by pediatricians trained in advanced airway management and life support. MATERIALS AND METHODS: Midazolam and ketamine were administered consecutively by intravenous route under cardiorespiratory monitoring for painful procedures of pediatric hematology. RESULTS: A total of 115 patients had 237 sedoanalgesia sessions. Sedation time was 24.02±23.37 s and sedation success was 92.5% (Ramsay scores of ≥5). Patient satisfaction was high. The recovery time was 28.81±14.4 min. Although statistically significant (p<0.01) increases in systolic and diastolic blood pressure, heart rate, and respiratory rate were observed without clinical importance, they improved without any intervention. No severe adverse events were observed. CONCLUSION: Sedoanalgesia with intravenous midazolam and ketamine for pediatric hematology and oncology patients' painful minor invasive procedures performed in an optimally equipped setting outside the operating room by pediatricians trained and certificated in advanced airway management and life support is effective and safe.

Screening survivors of childhood acute lymphoblastic leukemia for obesity, metabolic syndrome, and insulin resistance.

Acute lymphoblastic leukemia (ALL) survivors were screened for risk factors of cardiovascular disease. Forty-four ALL survivors in first remission were enrolled. Twenty-six also received 12-18 Gy cranial radiotherapy (RT). Patients' body mass indexes (BMIs) at dignosis and during the study were compared. Metabolic syndrome (MS) evaluation was performed in patients, parents, and siblings older than 6 years. Homeostasis Model Assessment (HOMA) index of the survivors was also calculated. In survivors with impaired fasting glucose levels, oral glucose tolerance test (OGTT) was performed. Thyroid functions and IGF-1 and/or IGFBP-3 levels of the survivors who received cranial RT were evaluated. Median age of the survivors was 11.5 years (6-23). At diagnosis, mean BMI percentile was 46.7 (3-95) and mean z-score was -0.09 ± 1.14; during the study, these values rose to 71.1 ± 25.6 (3-100) and 0.8 ± 0.94, respectively (P < .001). One patient (2.2%) and nine survivors (20%) were obese at diagnosis and during the study, respectively (P = .005). Survivors had significantly higher BMI percentile and BMI z-score compared to their siblings (P = .006 and P = .011, respectively). The study group was small and we could not show a correlation of the patients' obesity with RT, thyroid functions, IGF-1, and IGFBP-3 levels. In three survivors (6.8%), there was MS. Maternal and paternal MS was not found as a risk factor for MS of the survivors (P = .1, P = .5, respectively). The HOMA index revealed insulin resistance (IR) in 12 (27.2%) of the survivors, whereas OGTT revealed abnormal glucose regulation and/or IR in four. As a conclusion, ALL survivors have high risk for obesity and MS.

Management of autoimmune hemolytic anemia in children and adolescents: A single center experience.

OBJECTIVE: To present and discuss the treatment of autoimmune hemolytic anemia (AIHA). METHODS: The medical records of all patients (n=19) diagnosed in a tertiary hematology center between 1999 and 2010 were retrospectively reviewed. RESULTS: Median age at diagnosis of AIHA was 5 years (range: 4 months-17 years). In all, 13 patients had primary (idiopathic) AIHA, whereas 2 had primary Evans Syndrome (ES), 2 had autoimmune lymphoproliferative syndrome (ALPS)+ES, and 1 had Wiskott-Aldrich syndrome (WAS)+AIHA. Among the 13 primary idiopathic AIHA patients, 9 recovered following a 4-8-week course of prednisolone treatment without relapses, whereas 3 patients required a longer course of prednisolone. One AIHA patient that was very resistant to prednisolone recovered after cyclosporine A was added to the treatment. All patients with primary idiopathic AIHA were in remission for a median of 3 years (range: 4 months-10 years) at the time this manuscript was written. Among the patients with primary ES, 2 had relapses similar to the ALPS patients. Splenectomy was performed in 1 primary ES patient, who at the time this report was written was also in remission. One ALPS patient required the addition of mycophenolate mofetil due to prednisolone resistance. The WAS patient was treatment resistant and died due to septicemia. CONCLUSION: Primary AIHA in pediatric patients generally has an acute onset and good response to corticosteroids. Primary or secondary ES has a chronic or relapsing course, and treatment may require other immunosuppressive agents in addition to corticosteroids. Complications of splenectomy must not be underestimated in patients with underlying immunodeficiency. AIHA often causes considerable morbidity and mortality in WAS.