Case Report: MYH9-related disease caused by Ala44Pro mutation in a child with a previous diagnosis of chronic immune thrombocytopenia.

MYH9-related disease, a rare autosomal dominant platelet disorder characterized by thrombocytopenia, giant platelets, and leukocyte inclusion bodies, may mimic immune thrombocytopenia in children unless suspected and carefully excluded. Here, we present a case involving a three-year-old girl with mild bleeding symptoms since infancy, previously diagnosed with chronic immune thrombocytopenia. The patient exhibited isolated thrombocytopenia and lacked any family history of thrombocytopenia, hearing impairment, or renal failure. Examination of peripheral blood smears via light microscopy revealed significant platelet macrocytosis with giant platelets and basophilic Döhle-like bodies in the neutrophils. Subsequent sequencing analysis of MYH9 gene identified a p.Ala44Pro mutation. Throughout a six-year follow-up period, the patient's condition remained stable. Our report underscores the significance of identifying leukocyte inclusion bodies in peripheral blood smears and considering MYH9-related diseases, even in instances of chronic macrothrombocytopenia devoid of familial history or non-hematological manifestations.

TBI, etoposide, and cyclophosphamide conditioning for intermediate-risk relapsed childhood acute lymphoblastic leukemia.

BACKGROUND: In children with intermediate-risk relapsed acute lymphoblastic leukemia (ALL), allogeneic hematopoietic stem cell transplantation (allo-HSCT) has markedly improved the outcome of patients with an unsatisfactory minimal residual disease (MRD) response. Total body irradiation (TBI), etoposide (ETP), and cyclophosphamide (CY) have been shown to be equivalent to or better than TBI + ETP for conditioning, so we hypothesized that even greater survival could be achieved due to recent advances in HSCT and supportive care. PROCEDURE: We prospectively analyzed the efficacy and safety of allo-HSCT with a unified conditioning regimen of TBI + ETP + CY in children with intermediate-risk relapsed ALL, based on MRD in the bone marrow after induction, from the Japanese Pediatric Leukemia/Lymphoma Study Group (JPLSG) ALL-R08-II nationwide cohort (UMIN000002025). RESULTS: Twenty patients with post-induction MRD ≥ 10-3 and two not evaluated for MRD underwent allo-HSCT. Engraftment was confirmed in all patients, and no transplantation-related mortality was observed. The 3-year event-free survival and overall survival rates after transplantation were 86.4% ± 7.3% and 95.5% ± 4.4%, respectively. CONCLUSION: Allo-HSCT based on post-induction MRD with TBI + ETP + CY conditioning was feasible in Japanese children with intermediate-risk relapsed ALL.

Case report: Thyroid storm in a three-year-old girl presenting with febrile status epilepticus and hypoglycemia.

Thyroid storm, though extremely rare in toddlers, requires prompt diagnosis and treatment because it can be fatal if left untreated. However, thyroid storm is not often considered in the differential diagnosis of a febrile convulsion due to its rarity in children. Herein, we report the case of a 3-year-old girl with thyroid storm who presented with febrile status epilepticus. Although the seizure was stopped by diazepam administration, her tachycardia and widened pulse pressure persisted, and severe hypoglycemia was observed. Based on the findings of thyromegaly, a history of excessive sweating and hyperactivity, and a family history of Graves' disease, she was eventually diagnosed with a thyroid storm. The patient was successfully treated with thiamazole, landiolol, hydrocortisone, and potassium iodide. Propranolol, a non-selective ß-blocker, has been used to manage tachycardia during thyroid storm. However, a cardio-selective ß1-blockers, landiolol hydrochloride, was used in our case to avoid worsening hypoglycemia. Febrile status epilepticus is one of the most common medical emergencies in childhood; it is necessary to rule out treatable underlying critical diseases such as septic meningitis and encephalitis. Thyroid storm should be considered in children presenting with prolonged febrile convulsion accompanied by findings that are not usually observed with febrile convulsions.

Phase 2 study of combination chemotherapy with bortezomib in children with relapsed and refractory acute lymphoblastic leukemia.

Treatment outcomes for children with relapsed and refractory acute lymphoblastic leukemia (R/R-ALL) remain poor, and the optimal induction therapy has not been determined. Bortezomib is a proteasome inhibitor that acts synergistically and additively with standard chemotherapy for ALL. We evaluated the efficacy and safety of combination chemotherapy with bortezomib in children with R/R-ALL. This single-arm, multicenter, phase 2 study was conducted in Japan between 2016 and 2020. Eligible patients were divided into two cohorts: a high-risk first-relapse cohort of untreated patients with high-risk first-relapsed ALL and an expansion cohort of patients with refractory ALL, including multiple relapses, relapse after allogeneic hematopoietic cell transplantation, and induction failure. All patients received a single course of chemotherapy as induction therapy. Sixteen patients (10 in the high-risk first-relapse cohort, six in the expansion cohort) were evaluable. The overall remission rate after induction therapy was 60% in the high-risk first-relapse cohort and 16.7% in the expansion cohort. All patients had minimal residual disease. Adverse events were acceptable except for interstitial lung disease and hypoxia in a patient in the expansion cohort, but addition of bortezomib to conventional chemotherapy did not produce obvious improvement in children with R/R-ALL.

Improved Antitumor Effect of NK Cells Activated by Neutrophils in a Bone Marrow Transplant Model.

The licensing process mediated by inhibitory receptors of the Ly49 C-type lectin superfamily that recognizes self-major histocompatibility complex (MHC) class I in mice is essential for the proper antitumor function of natural killer (NK) cells. Several models for NK cell licensing can be exploited for adoptive immunotherapy for cancer. However, the appropriate adoptive transfer setting to induce efficient graft versus tumor/leukemia effects remains elusive, especially after hematopoietic stem cell transplantation (HSCT). In our previous experiment, we showed that intraperitoneal neutrophil administration with their corresponding NK receptor ligand-activated NK cells using congenic mice without HSCT. In this experiment, we demonstrate enhanced antitumor effects of licensed NK cells induced by weekly intraperitoneal injections of irradiated neutrophil-enriched peripheral blood mononuclear cells (PBMNCs) in recipient mice bearing lymphoma. Bone marrow transplantation was performed using BALB/c mice (H-2d) as the recipient and B10 mice (H-2b) as the donor. The tumor was A20, a BALB/c-derived lymphoma cell line, which was injected subcutaneously into the recipient at the same time as the HSCT. Acute graft versus host disease was not exacerbated in this murine MHC class I mismatched HSCT setting. The intraperitoneal injection of PBMNCs activated a transient licensing of NK subsets expressed Ly49G2, its corresponding NK receptor ligand to H-2d, and reduced A20 tumor growth in the recipient after HSCT. Pathological examination revealed that increased donor-oriented NK1.1+NK cells migrated into the recipient tumors, depending on neutrophil counts in the administered PBMNCs. Collectively, our data reveal a pivotal role of neutrophils in promoting NK cell effector functions and adoptive immunotherapy for cancer.

Successful hematopoietic stem cell transplantation for two patients with relapse of intrachromosomal amplification of chromosome 21-positive B-cell precursor acute lymphoblastic leukemia.

In children with relapsed acute lymphoblastic leukemia (ALL), it is essential to identify patients in need of treatment intensification. Minimal residual disease (MRD)-based treatment stratification resulted in excellent survival in children with late relapsed B-cell precursor (BCP)-ALL. Chemotherapy alone produced a favorable outcome in patients with negative MRD after induction. The genetic abnormality also plays an important role in determining the prognosis and stratification for treatment. Intrachromosomal amplification of chromosome 21 (iAMP21) is associated with a poor outcome and a high risk for relapse, and there is no standard treatment after relapse. Herein, we present two patients with relapsed iAMP21-positive ALL who were successfully treated by cord blood transplantation (CBT). Although both patients had late bone marrow relapse and favorable MRD response, CBT was performed due to iAMP21 positive. Patients 1 and 2 have been in remission post-CBT for 15 and 45 months, respectively. Patients with relapsed iAMP21-positive ALL may be considered for stem cell transplantation even in late relapses and favorable MRD response.

Autonomic Nervous System in Preterm Very Low Birth Weight Neonates with Intraventricular Hemorrhage.

OBJECTIVE: Heart rate variability (HRV) indicates cardiac autonomic nerve activity and is influenced by brain damage during the neonatal period. We aimed to determine whether a correlation exists between the HRV of extremely preterm neonates and neurodevelopmental test scores. STUDY DESIGN: Electrocardiogram data of neonates were assessed and HRV patterns in extremely preterm neonates with severe intraventricular hemorrhage (IVH; n = 6) and those with no/mild IVH (n = 28) were compared. We analyzed the relationship between HRV and neurodevelopmental outcomes at 18 months (n = 21) and 3 years (n = 23) in extremely preterm neonates. RESULTS: HRV was significantly associated with IVH severity in extremely preterm neonates (p < 0.05). Neonates with severe IVH exhibited increased HR and decreased mean R-to-R interval (NN) compared with neonates with no/mild IVH. HRV parameters significantly decreased in the severe IVH group, but not in the no/mild IVH group, suggesting that both sympathetic and parasympathetic activities decreased in neonates with severe IVH. Additionally, decreased HR and increased NN were significantly related to impaired neurodevelopmental outcomes in the no/mild IVH group at corrected ages of 18 months and 3 years, respectively (all p < 0.05). CONCLUSION: HRV was significantly associated with IVH severity and neurodevelopmental outcome in extremely preterm neonates. HRV can distinguish extremely preterm neonates who subsequently had severe IVH from those who had no/low-grade IVH. HRV may identify extremely preterm neonates needing adjuvant neuroprotective interventions. These findings warrant further investigation in a larger population of extremely preterm neonates. KEY POINTS: · HRV was associated with IVH severity.. · HRV can predict subsequent severe IVH in extremely preterm neonates.. · HRV are predictive of neurodevelopmental outcomes in extremely premature neonates with low-grade IVH..

Nafamostat mesylate prevents metastasis and dissemination of neuroblastoma through vascular endothelial growth factor inhibition.

Neuroblastoma is a highly malignant disease with a poor prognosis and few treatment options. Despite conventional chemotherapy for neuroblastoma, resistance, invasiveness, and metastatic mobility limit the treatment efficacy. Therefore, it is necessary to develop new strategies for treating neuroblastoma. The present study aimed to evaluate the anticancer effects of nafamostat mesylate, a previously known serine protease inhibitor, on neuroblastoma cells. Effects of nafamostat mesylate on neuroblastoma cell migration and proliferation were analyzed by wound healing assay and WST-8 assay, respectively. To elucidate the mechanisms underlying the effects of nafamostat mesylate on neuroblastoma, the expression levels of NF-κB were measured via western blotting, and the production of the cytokine vascular endothelial growth factor (VEGF) in the cell culture supernatants was determined via ELISA. In addition, a mouse model of hematogenous metastasis was used to investigate the effects of nafamostat mesylate on neuroblastoma. It was determined that nafamostat mesylate significantly inhibited migration and invasion of Neuro-2a cells, but it had no effect on cell proliferation at 24 h after treatment. Exposure of Neuro-2a cells to nafamostat mesylate resulted in decreased vascular endothelial growth factor production, which could be a pivotal mechanism underlying the inhibitory effects of neuroblastoma metastasis. The results of the present study suggest that nafamostat mesylate may be an effective treatment against neuroblastoma invasion and metastasis.

The first Japanese biobank of patient-derived pediatric acute lymphoblastic leukemia xenograft models.

A lack of practical resources in Japan has limited preclinical discovery and testing of therapies for pediatric relapsed and refractory acute lymphoblastic leukemia (ALL), which has poor outcomes. Here, we established 57 patient-derived xenografts (PDXs) in NOD.Cg-Prkdcscid ll2rgtm1Sug /ShiJic (NOG) mice and created a biobank by preserving PDX cells including three extramedullary relapsed ALL PDXs. We demonstrated that our PDX mice and PDX cells mimicked the biological features of relapsed ALL and that PDX models reproduced treatment-mediated clonal selection. Our PDX biobank is a useful scientific resource for capturing drug sensitivity features of pediatric patients with ALL, providing an essential tool for the development of targeted therapies.

Donor-derived M2 macrophages attenuate GVHD after allogeneic hematopoietic stem cell transplantation.

INTRODUCTION: Graft-versus-host disease (GVHD) is frequent and fatal complication following allogeneic hematopoietic stem cell transplantation (HSCT) and characteristically involves skin, gut, and liver. Macrophages promote tissue regeneration and mediate immunomodulation. Macrophages are divided into two different phenotypes, classically activated M1 (pro-inflammatory or immune-reactive macrophages) and alternatively activated M2 (anti-inflammatory or immune-suppressive macrophages). The anti-inflammatory effect of M2 macrophage led us to test its effect in the pathophysiology of GVHD. METHODS: GVHD was induced in lethally irradiated BALB/c mice. M2 macrophages derived from donor bone marrow (BM) were administered intravenously, while controls received donor BM-mononuclear cells and splenocytes. Animals were monitored for clinical GVHD and analyzed. RESULTS: We confirmed that administering donor BM-derived M2 macrophages attenuated GVHD severity and prolonged survival after HSCT. Moreover, donor BM-derived M2 macrophages significantly suppressed donor T cell proliferation by cell-to-cell contact in vitro. CONCLUSIONS: We showed the protective effects of donor-derived M2 macrophages on GVHD and improved survival in a model of HSCT. Our data suggest that donor-derived M2 macrophages offer the potential for cell-based therapy to treat GVHD.

Primary cytomegalovirus infection during pregnancy and congenital infection: a population-based, mother-child, prospective cohort study.

OBJECTIVE: This study assessed maternal cytomegalovirus antibodies, and the occurrence of primary and congenital cytomegalovirus infections, and risk factors of congenital infection after a maternal primary infection. STUDY DESIGN: We included 19,435 pregnant women in Japan, who were tested for serum cytomegalovirus antibodies before 20 gestational weeks. Immunoglobulin (Ig) G avidity was evaluated in women with both IgG and IgM antibodies; tests were repeated at ≥28 gestational weeks among women without IgG and IgM antibodies. RESULT: Primary and congenital infections were 162 and 23 cases, respectively. The risk ratios for congenital infection were 8.18 (95% confidence interval: 2.44-27.40) in teenage versus older women, and 2.25 (95% confidence interval: 1.28-3.94) in parity ≥ 2 versus parity ≤ 1. Of 22 live birth congenital infection cases, three had abnormal neurological findings. CONCLUSION: We demonstrated teenage and parity ≥ 2 pregnant women as risk factors of post-primary congenital infection.

Case Report: Effects of Secondary Hyperparathyroidism Treatment on Improvement of Juvenile Nephronophthisis-Induced Pancytopenia and Myelofibrosis.

Secondary hyperparathyroidism (HPT) is a common complication of end-stage renal disease (ESRD) and may be an important precipitating factor for the development of myelofibrosis. However, there have been only a few reports on myelofibrosis caused by secondary HPT in children. We describe a case of a 15-year-old boy with myelofibrosis due to secondary HPT who was successfully treated with hemodialysis, erythropoietin, phosphate binders, and activated vitamin D agents. The patient had no past medical history and had been admitted to the hospital for abdominal pain. Routine blood examination revealed pancytopenia combined with renal impairment. Hyperphosphatemia, decreased 1,25-dehydroxyvitamin D, decreased serum calcium, and increased parathyroid hormone (PTH) levels were observed. Bone marrow biopsy confirmed myelofibrosis and renal biopsy revealed nephronophthisis (NPHP). The possibility of renal osteodystrophy and myelofibrosis due to secondary HPT was considered. Hemodialysis and erythropoietin were initiated and combined therapy with a phosphate binder and an active vitamin D agent achieved greater reduction of PTH levels, along with improvement of pancytopenia. As medical treatment for secondary HPT can lead to a reversal of myelofibrosis and avoid parathyroidectomy in children, prompt recognition of this condition has major implications for treatment. Therefore, despite its rarity, pediatricians should consider myelofibrosis due to secondary HPT as a cause of pancytopenia in patients with chronic kidney disease.

Anti-tumor effect of Huaier extract against neuroblastoma cells in vitro.

Huaier extract, the main active constituent proteoglycan, has anti-tumor activity in various experimental and clinical settings. However, the potential anti-neuroblastoma and associated mechanisms have not been investigated. Therefore, in this study, we aimed to elucidate the potential role of Huaier extract in 3 human neuroblastoma cell lines. Our study demonstrated that incubation with Huaier extract resulted in a marked decrease in cell viability in a dose-dependent manner. Huaier extract induced cell cycle arrest at G0/G1 phase in neuroblastoma and decreased the cell cycle related protein expression of cyclin D3. Western blotting analysis also showed that Huaier extract induced neuroblastoma cell apoptosis and autophagy. Signaling analysis indicated that Huaier extract suppressed the MEK/ERK and mTOR signaling pathways simultaneously. In conclusion, we verify that Huaier extract causes cell proliferation inhibition, apoptosis, autophagy, and cell cycle arrest in G0/G1 phase via MEK/ERK and mTOR signaling. Huaier extract may act as a complementary agent for treating neuroblastoma.

Report of 2 Pediatric Cases With Li-Fraumeni Syndrome Related Malignancy in a Family.

Li-Fraumeni syndrome (LFS) is a rare inherited disease characterized by a high and early-onset cancer risk. A cancer surveillance program is important to reduce cancer-related morbidity and mortality in individuals with LFS. We report 2 pediatric cases with LFS-related malignancy in a family. Eight-year-old elder brother was diagnosed with adrenocortical carcinoma and was found to have a heterozygous missense germline mutation c.736A>G: p.Met246Val in the TP53 gene. Cancer screening led to the diagnosis of rhabdomyosarcoma at a curable stage in his 2-year-old younger brother. Comprehensive surveillance resulted in early tumor detection and improved survival.

Huaier Extract Induces Apoptosis in Hepatoblastoma Cells Via the MEK/ERK Signaling Pathway.

BACKGROUND/AIM: Huaier extract, whose main active constituent is the proteoglycan, has anti-tumor activity in several types of malignancies. In this study, we aimed to elucidate the effect of Huaier extract in hepatoblastoma cells. MATERIALS AND METHODS: The effect of Huaier extract on the proliferation of human hepatoblastoma cell lines HepG2 and HuH-6, was examined. RESULTS: Incubation with Huaier extract resulted in a marked, dose-dependent decrease in hepatoblastoma cell viability. Huaier extract induced S phase arrest in hepatoblastoma cells and upregulated the expression of the cell cycle related proteins cyclin D1 and cyclin D3. It also induced apoptosis in hepatoblastoma cells. Additionally, it significantly suppressed the activity of p-ERK and p-MEK. CONCLUSION: Huaier extract inhibits proliferation, and induces cell apoptosis and cell cycle arrest via the MEK-ERK pathway in hepatoblastoma cells. Huaier extract may act as a complementary agent for treating hepatoblastoma.

Refractory Ileal Perforations in a Cytomegalovirus-Infected Premature Neonate Resolved After Ganciclovir Therapy.

Severe neonatal gastrointestinal diseases such as necrotizing enterocolitis or spontaneous intestinal perforation are potentially lethal conditions which predominantly occur in preterm infants. Cytomegalovirus (CMV), which is known to cause congenital and acquired infections in the newborns, has also been implicated in such severe gastrointestinal diseases in premature infants. However, the pathogenic role of CMV and effect of antiviral therapy in severe gastrointestinal disease in premature neonates is currently unclear. We present an infant, born at 26-weeks' gestation, presented with progressive dyspepsia and abdominal distention after the closure of the symptomatic patent ductus arteriosus at the day of life (DOL) 4, requiring the emergent surgery for ileal perforation at the DOL8. After the surgery, abdominal symptoms persisted and the second emergent surgery was performed for the recurrent ileal perforation at DOL17. Even then the abdominal symptoms prolonged and pathological examination in the affected intestine at the second surgery showed CMV inclusion body. Immunoreactivity for CMV antigen was detected in the specimen at the first surgery on DOL8. Blood and urinary CMV-DNA were detected at DOL28. CMV-DNA was also detected in the dried umbilical cord which was obtained within a week from birth. A 6-week course of intravenous ganciclovir (12 mg/kg/day) was started at DOL34 and then symptoms resolved along with decreasing blood CMV-DNA. Pathological findings characteristic of CMV were not detected in the resection specimen at the ileostomy closure at DOL94. These observations indicate that anti-CMV therapy may be beneficial for some premature infants with severe CMV-associated gastrointestinal diseases and warrants further studies focusing on pathogenic role, diagnosis, treatment and prevention of this underrecognized etiology of severe gastrointestinal diseases particularly in premature neonates.

FLEND (nelarabine, fludarabine, and etoposide) for relapsed T-cell acute lymphoblastic leukemia in children: a report from Japan Children's Cancer Group.

Nelarabine is a key drug for T-cell acute lymphoblastic leukemia (T-ALL). Fludarabine and etoposide might have synergistic effect with nelarabine by inhibiting ribonucleotide reductase and by preparing cell cycle for G1/S phase, respectively. We had started phase 1/2 multicenter clinical trial of combination chemotherapy consisted of nelarabine, fludarabine, and etoposide (FLEND therapy) for children with relapsed/refractory T-ALL which has been conducted since October 2011. Although we could not complete this trial because of recruitment difficulties, we treated five children with first-relapsed T-ALL which were enrolled in the phase 1 dose escalation study of fludarabine and etoposide with nelarabine. No dose-limiting toxicity occurred, and frequent grade 3-4 toxicity was hematological toxicity and febrile neutropenia, as expected. There was no neurotoxicity. All 2 patients who received the target dose of FLEND, in which nelarabine (650 mg/m2), fludarabine (30 mg/m2), and etoposide (100 mg/m2) were administered for 5 consecutive days, were induced to complete remission. We concluded that FLEND might be safe and one of the promising combination chemotherapies to relapsed/refractory T-ALL.

Prevalence of germline GATA2 and SAMD9/9L variants in paediatric haematological disorders with monosomy 7.

Monosomy 7 (-7) occurs in various types of paediatric myeloid disorders and has a poor prognosis. Recent studies have demonstrated that patients with germline gain-of-function SAMD9/9L variants and loss-of-function GATA2 variants are prone to developing myelodysplastic syndrome (MDS) associated with -7. However, the prevalence of the genetic variants among paediatric haematologic disorders with -7 is unknown. The present study screened germline variants of GATA2 and SAMD9/9L in 25 patients with various types of paediatric haematological disorders associated with -7. The diagnoses of the 25 patients included MDS (n = 10), acute myeloid leukaemia (AML) and myeloid sarcomas (n = 9), juvenile myelomonocytic leukaemia (n = 3) and other disorders (n = 3). Seven patients with a germline pathogenic GATA2 variant were found. For SAMD9/9L screening, next-generation sequencing was used to detect low-abundance variants and found four novel germline variants. Functional analysis revealed that three out of the four variants showed growth-restricting capacity in vitro and thus, were judged to be pathogenic. Cases with GATA2 mutation tended to be older, compared to those with SAMD9/9L mutations. In conclusion, GATA2 and SAMD9/9L were sequenced in 25 patients with paediatric haematologic disorders associated with -7, and 40% of them were found to have some pathogenic germline variants in the three genes.