Six Years of Disease-free Survival After a Second Cord Blood Transplantation for Recurrent Acute Lymphocytic Leukemia in a Child With Down Syndrome.

Outcomes are extremely poor in Down syndrome-associated acute lymphocytic leukemia, particularly in recurrent cases. A 2-year-old boy with Down syndrome-associated acute lymphocytic leukemia achieved complete remission after standard chemotherapy. However, he experienced recurrence twice in the bone marrow and central nervous system. Salvage treatments included whole-brain/whole-spine irradiation. Thereafter, the patient received a second cord blood transplantation after the reduced-intensity conditioning. The graft was characterized by killer cell immunoglobulin-like receptor ligands mismatch. The patient has subsequently survived for 6.5 years without recurrence. We speculate that killer cell immunoglobulin-like receptor ligand-mismatched cord blood transplantation enhanced the graft-versus-leukemia effect through natural killer cells, and conferred long-term remission.

Eculizumab treatment in paediatric patients diagnosed with aHUS after haematopoietic stem cell transplantation: a HSCT-TMA case series from Japanese aHUS post-marketing surveillance.

Haematopoietic stem-cell transplantation (HSCT)-associated thrombotic microangiopathy (HSCT-TMA) is a serious complication with high mortality. Accumulating evidence suggests that complement dysregulation is potentially involved in the development of HSCT-TMA. We retrospectively analysed the clinical characteristics and outcomes of thirteen paediatric patients who were diagnosed with atypical haemolytic uremic syndrome and treated with eculizumab to manage HSCT-TMA during post-marketing surveillance in Japan. The median time from HSCT to TMA was 31 days (Interquartile range, IQR;21-58) and the median doses of eculizumab was three (IQR;2-5). Seven patients (54%) were alive at the last follow-up while six died due to complications related to HSCT. Six of seven survivors initiated eculizumab after insufficient response to plasma therapy. Following eculizumab treatment, median platelet counts and LDH levels in all survivors significantly improved and renal function improved in 4/7 patients. All survivors possessed potential risk factors of complement overactivation. During the follow-up period after eculizumab discontinuation (median;111.5 days, IQR;95-555), no TMA recurrence was observed. In this analysis, eculizumab showed benefit in over half of this paediatric patient population. Ongoing clinical studies are expected to optimize the treatment regimen of terminal complement pathway inhibitor, and it may become a therapeutic option for paediatric HSCT-TMA in the future.

Variant spectrum of PIEZO1 and KCNN4 in Japanese patients with dehydrated hereditary stomatocytosis.

Hereditary stomatocytosis (HSt) is a type of congenital hemolytic anemia caused by abnormally increased cation permeability of erythrocyte membranes. Dehydrated HSt (DHSt) is the most common subtype of HSt and is diagnosed based on clinical and laboratory findings related to erythrocytes. PIEZO1 and KCNN4 have been recognized as causative genes, and many related variants have been reported. We analyzed the genomic background of 23 patients from 20 Japanese families suspected of having DHSt using a target capture sequence and identified pathogenic/likely pathogenic variants of PIEZO1 or KCNN4 in 12 families.

Breakpoint analysis for cytogenetically balanced translocation revealed unexpected complex structural abnormalities and suggested the position effect for MEF2C.

Many disease-causing genes have been identified by determining the breakpoints of balanced chromosomal translocations. Recent progress in genomic analysis has accelerated the analysis of chromosomal translocation-breakpoints at the nucleotide level. Using a long-read whole-genome sequence, we analyzed the breakpoints of the cytogenetically balanced chromosomal translocation t(5;15)(q21;26.3), which was confirmed to be of de novo origin, in a patient with a neurodevelopmental disorder. The results showed complex rearrangements with seven fragments consisting of five breakpoint-junctions (BJs). Four of the five BJs showed microhomologies of 1-3-bp, and only one BJ displayed a signature of blunt-end ligation, indicating chromothripsis as the underlying mechanism. Although the BJs did not disrupt any disease-causing gene, the clinical features of the patient were compatible with MEF2C haploinsufficiency syndrome. Complex rearrangements were located approximately 2.5-Mb downstream of MEF2C. Therefore, position effects were considered the mechanism of the occurrence of MEF2C haploinsufficiency syndrome.

Cord Blood Transplantation in 2 Infants Presenting Monosomy 7 Clonal Hematopoiesis: SAMD9 / SAMD9L Germline Mutation.

Recently, germline mutations in SAMD9 and SAMD9L were increasingly found in children with monosomy 7. We report the outcomes in 2 infants with the SAMD9/SAMD9L variant, who presented with anemia and thrombocytopenia (patient 1), and neutropenia and nonsymptomatic white-matter-encephalopathy (patient 2). Both patients received cord blood transplantation and experienced critical post-cord blood transplantation adverse events; patients 1 and 2 developed fulminant engraftment syndrome and life-threatening graft-versus-host disease, respectively. Of note, selective loss of chromosome 7 in bone marrow-derived CD34 + cells was inferred.

Long-read sequence analysis for clustered genomic copy number aberrations revealed architectures of intricately intertwined rearrangements.

Most chromosomal aberrations revealed by chromosomal microarray testing (CMA) are simple; however, very complex chromosomal structural rearrangements can also be found. Although the mechanism of structural rearrangements has been gradually revealed, not all mechanisms have been elucidated. We analyzed the breakpoint-junctions (BJs) of two or more clustered copy number variations (CNVs) in the same chromosome arms to understand their conformation and the mechanism of complex structural rearrangements. Combining CMA with long-read whole-genome sequencing (WGS) analysis, we successfully determined all BJs for the clustered CNVs identified in four patients. Multiple CNVs were intricately intertwined with each other, and clustered CNVs in four patients were involved in global complex chromosomal rearrangements. The BJs of two clustered deletions identified in two patients showed microhomologies, and their characteristics were explained by chromothripsis. In contrast, the BJs in the other two patients, who showed clustered deletions and duplications, consisted of blunt-end and nontemplated insertions. These findings could be explained only by alternative nonhomologous end-joining, a mechanism related to polymerase theta. All the patients had at least one inverted segment. Three patients showed cryptic aberrations involving a disruption and a deletion/duplication, which were not detected by CMA but were first identified by WGS. This result suggested that complex rearrangements should be considered if clustered CNVs are observed in the same chromosome arms. Because CMA has potential limitations in genotype-phenotype correlation analysis, a more detailed analysis by whole genome examination is recommended in cases of suspected complex structural aberrations.

Interstitial microdeletions of 3q26.2q26.31 in two patients with neurodevelopmental delay and distinctive features.

Interstitial microdeletions in the long arm of chromosome 3 are rare. In this study, we identified two patients with approximately 5-Mb overlapping deletions in the 3q26.2q26.31 region. Both patients showed neurodevelopmental delays, congenital heart defects, and distinctive facial features. One of them showed growth deficiency and brain abnormalities, as shown on a magnetic resonance imaging scan. Haploinsufficiency of NLGN1 and FNDC3B present in the common deletion region was considered to be responsible for neurodevelopmental delay and the distinctive features, respectively. The possibility of unmasked variants in PLD1 was considered and analyzed, but no possible pathogenic variant was found, and the mechanism of the congenital heart defects observed in the patients is unknown. Because 3q26.2q26.31 deletions are rare, more information is required to establish genotype-phenotype correlations associated with microdeletions in this region.

Case Report: Successful Treatment With Anti-C5 Monoclonal Antibody in a Japanese Adolescent Who Developed Thrombotic Microangiopathy After Autologous Bone Marrow Transplantation for Malignant Lymphoma.

Background: Transplant-associated thrombotic microangiopathy (TA-TMA) is a serious complication of bone marrow transplantation (BMT). Recently, abnormalities in the complement system have been identified in the pathogenesis of TA-TMA, and there are series of reports stating that anti-C5 monoclonal antibody (eculizumab) is effective in patients with high levels of the membrane attack complex (C5b-9). Case Presentation: A 12-year-old boy underwent autologous BMT after receiving high-dose chemotherapy for malignant lymphoma. The patient was engrafted on day 19 after transplantation; however, hemolytic anemia and non-immune thrombocytopenia persisted, and haptoglobin decreased on day 46. Moreover, on day 83, the patient developed pulmonary hemorrhage, hypertension, severe proteinuria, hematuria, and acute kidney injury (AKI). Pulmonary bleeding stopped with daily platelet transfusion and hemostatic agents, but reappeared on day 100. Based on the presence of destruction of red blood cells, elevated lactate dehydrogenase levels, negative direct and indirect Coombs tests, normal ADAMTS13 levels, hemolytic anemia, non-immune thrombocytopenia, and AKI, the patient was diagnosed with systemic TA-TMA and we initiated plasma exchange (PE) and continuous hemodialysis for AKI. High C5b-9 levels were identified at the start of the series of PE, therefore we decided to administer eculizumab. After three courses of eculizumab, no pulmonary hemorrhage was observed, and anemia, thrombocytopenia, renal dysfunction, hematuria, and proteinuria all tended to improve. Three years after transplantation, the patient is alive and does not require eculizumab. Discussion: Eculizumab is a humanized monoclonal antibody that binds complement protein C5, preventing cleavage C5 and the formation of C5b-9. In this case, TA-TMA could not be controlled with PE alone. We therefore decided to use eculizumab relatively early based on the high C5b-9 level and could resolve the momentum of TA-TMA. Conclusion: In previous reports, TA-TMA typically occurred in early post-allogeneic BMT of patients with lymphoma or in post-autologous BMT of patients with neuroblastoma and was treated with eculizumab. We here reported that eculizumab could be successful treatment for TA-TMA in post-autologous BMT of patient with lymphoma.

Intracranial Bleeding as a Major Problem Among Neonates With Disseminated Intravascular Coagulation After the Introduction of Recombinant Thrombomodulin.

A standard treatment for disseminated intravascular coagulation in neonates has not yet been established. We analyzed the outcomes of 23 neonates who developed disseminated intravascular coagulation due to infection or asphyxia between 2004 and 2017. The overall survival rate was 95.7% on day 28 after anticoagulant therapy. In contrast, the bleeding-free survival rate was 69.6% (95% confidence interval, 53.1%-91.2%). Of the 6 neonates with intracranial bleeding, 2 developed neurological sequelae. The current study showed that intracranial bleeding remained a major problem in the early 2000s, despite the introduction of a new anticoagulant drug, recombinant thrombomodulin, at our institution since 2009.

Multiple Immunofluorescence Imaging Analysis Reveals Differential Expression of Disialogangliosides GD3 and GD2 in Neuroblastomas.

BACKGROUND: Peripheral neuroblastic tumors (pNTs) are the most common childhood extracranial solid tumors. There are several therapeutic strategies targeting disialoganglioside GD2. Disialoganglioside GD3 has become a potential target. However, the mechanism by which pNTs express GD3 and GD2 remains unclear. We investigated the combined expression status of GD3 and GD2 in pNTs and delineated their clinicopathological values. METHODS: GD3 and GD2 expression was examined in pNT tissue samples (n = 35) using immunohistochemistry and multiple immunofluorescence imaging. RESULTS: GD3 and GD2 expression was positive in 32/35 and 25/35 samples, respectively. Combinatorial analysis of GD3 and GD2 expression in neuroblastoma showed that both were heterogeneously expressed from cell to cell. There were higher numbers of GD3-positive and GD2-negative cells in the low-risk group than in the intermediate-risk (P = 0.014) and high-risk (P = 0.009) groups. Cases with high proportions of GD3-positive and GD2-negative cells were associated with the International Neuroblastoma Staging System stage (P = 0.004), Children's Oncology Group risk group (P = 0.001), and outcome (P = 0.019) and tended to have a higher overall survival rate. CONCLUSION: We demonstrated that neuroblastomas from low-risk patients included more GD3-positive and GD2-negative cells than those from high-risk patients. Clarifying the heterogeneity of neuroblastoma aids in better understanding the biological characteristics and clinical behavior.

Prognostic value of the revised International Prognostic Scoring System five-group cytogenetic abnormality classification for the outcome prediction of hematopoietic stem cell transplantation in pediatric myelodysplastic syndrome.

Cytogenetic abnormalities are a major risk factor for relapse after hematopoietic stem cell transplantation (HSCT) for myelodysplastic syndrome (MDS). We aimed to evaluate the value of the five-group cytogenetic classification according to the revised International Prognostic Scoring System (R-IPSS) for predicting the outcome after HSCT in pediatric patients with MDS. We retrospectively analyzed the Japanese registration data of 242 pediatric patients with MDS. According to the R-IPSS classification, 112 (45.5%) patients had good, 55 (22.7%) had intermediate, 64 (26.4%) had poor, and 11 (4.6%) had very poor cytogenetics. The 5-year overall survival (5yOS) was 72%, 69%, 59%, and 30% in the good, intermediate, poor, and very poor cytogenetic subgroups (p = 0.026), respectively. The very good, good, and intermediate subgroups were grouped into a "standard" subgroup and reclassified into three subgroups (standard, poor, and very poor). Patients with very poor risk had worse 5yOS (hazard ratio 2.17, 95% confidence interval (CI) 1.02-4.61; p = 0.04) and a much higher 5yCIR (hazard ratio 2.52, 95% CI 1.05-6.04; p = 0.04) than those of patients in the standard group in the multivariate analysis, indicating that very poor risk cytogenetic characteristics independently predicted worse outcome after HSCT in pediatric patients with MDS.

Incidence and outcomes of refractory immune thrombocytopenic purpura in children: a retrospective study in a single institution.

Treatment of children with refractory immune thrombocytopenic purpura (ITP) is challenging and poorly established. We retrospectively reviewed the clinical data of 87 patients under the age of 16 years who were diagnosed with ITP from April 1998 to March 2017 in our institution. Refractory ITP was defined as a platelet count of < 50 × 109/L at 14 days after receiving intravenous immunoglobulin (IVIG) and prednisolone. We presumed that there was a pathophysiological overlap between refractory ITP and refractory thrombocytopenia (RT): a subtype of refractory cytopenia of childhood (RCC). Immunosuppressive therapies including anti-thymocyte globulin and cyclosporine (CsA) have been adopted for children with RCC in Japan. Thus, from 2009 onwards, we changed the diagnosis from refractory ITP to RT and introduced CsA for refractory ITP/RT. Nine of 42 patients developed refractory ITP in the 1998-2008 group, who received conventional treatments such as IVIG and steroid therapy. Eight of 45 patients developed refractory ITP in the 2009-2017 group, who received CsA with or without IVIG therapy. The response rate at three years after diagnosis was significantly higher in the 2009-2017 group (98%) than in the 1998-2008 group (83%) (p = 0.019). In conclusion, our strategy of introducing CsA for refractory ITP/RT contributed to better outcomes.

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.