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ObjectiveCentral venous catheters (CVCs) have been essential devices for the treatment of children with hematological and oncological disorders. Only few studies investigated the complications and selections of different types of CVCs in these pediatric patients. This study aimed to compare risk factors for unplanned removal of two commonly used CVCs, i.e., peripherally inserted central catheters (PICCs) and tunneled CVCs, and propose better device selection for the patient.MethodsThis retrospective, single center cohort analysis was conducted on pediatric patients with hematological and oncological disorders inserted with either a PICC or a tunneled CVC.ResultsBetween January 1, 2013, and December 31, 2015, 89 patients inserted with tunneled CVCs (total 21,395 catheter-days) and 84 with PICCs (total 9,177 catheter-days) were followed up until the catheter removal. The median duration of catheterization was 88 days in PICCs and 186 days in tunneled CVCs (p = 1.24×10-9). PICCs at the 3-month cumulative incidence of catheter occlusion (5.2% vs. 0%, p = 4.08×10-3) and total unplanned removal (29.0% vs 7.0%, p = 0.0316) were significantly higher, whereas no significant difference was observed in the cumulative incidence of central line-associated bloodstream infection (11.8% vs. 2.3%, p = 0.664). Multivariable analysis identified younger age (ConclusionOur results suggest that tunnel CVCs would be a preferred device for children with hematological and oncological disorders requiring long-term, intensive treatment.

Key Points A total of 137 patients with JMML were analyzed using DREAM.We developed a robust DNA methylation test that is highly consistent with the array-based international consensus definition for JMML., Visual Abstract, Juvenile myelomonocytic leukemia (JMML) is a rare myelodysplastic/myeloproliferative neoplasm that develops during infancy and early childhood. The array-based international consensus definition of DNA methylation has recently classified patients with JMML into the following 3 groups: high (HM), intermediate (IM), and low methylation (LM). To develop a simple and robust methylation clinical test, 137 patients with JMML were analyzed using the Digital Restriction Enzyme Analysis of Methylation (DREAM), which is a next-generation sequencing–based methylation analysis. Unsupervised consensus clustering of the discovery cohort (n = 99) using DREAM data identified HM (HM_DREAM; n = 35) and LM subgroups (LM_DREAM; n = 64). Of the 98 cases that could be compared with the international consensus classification, 90 HM (n = 30) and LM (n = 60) cases had 100% concordance with DREAM clustering results. Of the remaining 8 cases comprising the IM group, 4 were classified as belonging to the HM_DREAM group and 4 to the LM_DREAM group. A machine-learning classifier was successfully constructed using a support vector machine (SVM), which divided the validation cohort (n = 38) into HM (HM_SVM, n = 18) and LM (LM_SVM; n = 20) groups. Patients with the HM_SVM profile had a significantly poorer 5-year overall survival rate than those with the LM_SVM profile. In conclusion, we developed a robust methylation test using DREAM for patients with JMML. This simple and straightforward test can be easily incorporated into diagnosis to generate a methylation classification for patients so they can receive risk-adapted treatment in the context of future clinical trials.

Objectives Patients with acquired aplastic anemia (AA) without HLA-matched sibling donors or aged >40 years receive immunosuppressive therapy (IST) with anti-thymocyte globulin (ATG). We investigated the relationship between plasma rabbit ATG (r-ATG) concentration and IST response. Methods From May 2012 to October 2017, 81 patients with severe AA who required initial IST were included. A 1:1 block randomization was employed for 2.5 and 3.5 mg/kg doses of r-ATG. Results No significant difference in response rates was observed between the 2.5 and 3.5 mg/kg groups (63% vs. 58%, P = .894). Median r-ATG concentrations on days 14 and 28 after IST were 15.2 (0.0-97.7) and 1.8 (0.0-74.9 µg/mL), respectively. According to r-ATG concentration, response rates were significantly higher in the group with higher r-ATG concentration than in those with lower r-ATG concentration (day 14, 88% vs. 52%; P = .006 and day 28, 79% vs. 46%; P = .005). In multivariate analysis, higher r-ATG concentrations at day 28 were independent predictors of favorable response to IST at 6 months (odds ratio, 0.29; 95% confidence interval, 0.09-0.93; P = .037). Conclusions The present data indicate that higher r-ATG concentration at day 28 resulted in improved IST response.

Aim: There may exist a gap between the current form of traditional Japanese Kampo pediatrics and Kampo pediatrics in the nineteenth century. This study aimed to elucidate changes in clinical practice by comparing the formulas used then and now. Methods: All formulas described in Yoyokasoku (In‐house Methods of Pediatrics [IMP], a Kampo classic published in 1885) and formulas used in a contemporary pediatric Kampo guideline were extracted. Then, a database with annotations to formulas was compiled. Results: In all, 387 formulas (274 excluding duplications) and 10 methods of moxibustion/surgical treatments were extracted from IMP. The total number of classical formulas described was 72. The proportion of classical formulas was significantly different among disease clusters (P, Version of Record online: 02 November 2020 ファイル公開：2021-11-02

Patients with primary refractory and relapsed neuroblastoma have a poor prognosis since safe and effective chemotherapies for these patients are currently limited. The development of new chemotherapy regimens for these patients is imperative to improve survival outcomes.We retrospectively analyzed 40 patients with refractory (n = 36) or relapsed (n = 4) neuroblastoma who received irinotecan, etoposide, and carboplatin (IREC) as a second-line treatment. We evaluated their therapeutic response and the toxicity of IREC. We also assessed the impact of UGT1A1 gene polymorphisms, which are involved in irinotecan metabolism, on outcomes and toxicity.A total of 112 cycles of IREC were administered to 40 patients with a median of 2 cycles per patient (range, 1-9). Six (15%) patients (UGT1A1 wild-type [n = 2] and heterozygous [n = 4]) showed objective responses, including partial response (n = 1), tumor shrinkage (n = 4), and improved findings on their MIBG scan (n = 1). Grade 4 neutropenia, grade 4 leukopenia, and grades 3-4 gastrointestinal toxicity were observed in 110 (98%), 88 (79%), and 3 (3%) cycles, respectively. There was no IREC-related mortality. Patients with UGT1A1 polymorphisms showed a higher frequency of grade 4 leukopenia, but these patients did not have increased treatment-related mortality or non-hematologic toxicity.IREC showed an objective response rate of 15% including 1 case with partial response. IREC was well tolerated regardless of UGT1A1 genotype. This study suggests that IREC is a promising second-line chemotherapy for refractory or relapsed neuroblastoma.

Pediatric solid tumors are a heterogeneous group of neoplasms with over 100 subtypes. Clinical and histopathological diagnosis remains challenging due to the overlapping morphological and immunohistochemical findings and the presence of atypical cases. To evaluate the potential utility of including RNA-sequencing (RNA-seq) in the diagnostic process, we performed RNA-seq in 47 patients with suspected pediatric sarcomas. Histopathologists specialized in pediatric cancer re-evaluated pathological specimens to reach a consensus diagnosis; 42 patients were diagnosed with known subtypes of solid tumors whereas 5 patients were diagnosed with undifferentiated sarcoma. RNA-seq analysis confirmed and refined consensus diagnoses and further identified diagnostic genetic variants in four of the five patients with undifferentiated sarcoma. Genetic lesions were detected in 23 patients, including the novel SMARCA4-THOP1 fusion gene and 22 conventional or recently reported genetic events. Unsupervised clustering analysis of the RNA-seq data identified a distinct cluster defined by the overexpression of rhabdomyosarcoma-associated genes including MYOG and CHRNG. These findings suggest that RNA-seq-based genetic analysis may aid in the diagnosis of suspected pediatric sarcomas, which would be useful for the development of stratified treatment strategies.

Pulmonary hypertension (PH) is associated with high morbidity in children undergoing hematopoietic stem cell transplantation (HSCT). However, owing to the lack of sequential echocardiography, the nature of the condition is not fully understood. This study was conducted to investigate whether routine echocardiography performed after HSCT could detect patients with PH at an earlier stage and elucidate the role of intervention using tadalafil. The study population comprised 93 consecutive children age18 years who underwent a total of 109 HSCTs. All patients underwent routine transthoracic echocardiography during HSCT. Four children (4%) with a median age of 4 years (range, 0.7 to 6 years) were found to have PH, and their median tricuspid regurgitation peak velocity (TRV) was 4.1 m/s (range, 3.5 to 4.2 m/s). PH was diagnosed at a median of 52 days (range, 21 to 118 days) after HSCT. Three of them were diagnosed with neuroblastoma, and 1 was diagnosed with infantile leukemia. One patient developed PH after autologous HSCT, and 3 received killer immunoglobulin-like receptor ligand-mismatched cord blood. Busulfan was used for conditioning in all patients, and the proportion of patients receiving this medication was significantly higher in the PH group compared with the non-PH group (100% versus 30%; P = .011). Three of the 4 patients had a durable response (TRV ≤2.8 m/s) at a median of 46 days (range, 14 to 79 days) after starting treatment with tadalafil. No patient experienced exacerbation of PH, and treatment was completed at median of 96 days (range, 46 to 212 days). Our data suggest that routine echocardiography monitoring after HSCT should be considered in children receiving busulfan, although the precise follow-up timing needs further study. In addition, safe and effective administration of tadalafil must be ensured by close monitoring.

Objective: The prognosis of adrenoleukodystrophy (ALD)with neurological involvement is generally dismal; however, allogeneic stem cell transplantation (SCT) is recognized as effective to stabilize or improve the clinical symptoms of ALD. Herein, we report the clinical outcomes of patients with ALD who consecutively underwent allogeneic stem cell transplantation with reduced intensity conditioning at our institution. Patients: Sixteen patients with ALD, who were symptomatic (n = 14) or presymptomatic (n = 2), received SCT from 2010 to 2016. The stem cell source was cord blood (n = 14), or bone marrow from a human leukocyte antigen identical sibling (n = 2). The conditioning regimen prior to transplantation was reduced intensity and consisted of fludarabine (125 mg/m2), melphalan (140 mg/m2) and low dose total body irradiation (TBI) of 4Gy (n = 15) or 3Gy (n = 1). Results: Primary engraftment was obtained in 11 patients, and 4 of the 5 patients who lost the primary graft received a second cord blood transplantation and were engrafted. Five years overall and event-free survival were 90.9% and 61.1% respectively, with a median of 45 months (range 16–91). Loes score stabilized or improved by 18 months after transplantation except for patients with internal capsule involvement. Conclusion: Allogeneic SCT with reduced intensity conditioning for patients with ALD was safely performed without major transplant-related complications even in symptomatic patients and neurological symptoms were stabilized after SCT in patients without internal capsule involvement. Keywords: Adrenoleukodystrophy, Allogeneic stem cell transplantation, Loes score, Very long chain fatty acid

We investigated the safety and efficacy of mycophenolate mofetil (MMF) in the prevention and treatment of graft-versus-host disease (GVHD) using a nationwide retrospective survey in Japanese children undergoing hematopoietic stem cell transplantation (HSCT). Overall, 141 children undergoing allogeneic HSCT for hematological malignancy (n = 84), non-malignancy (n = 52), and solid tumors (n = 5) were administered MMF orally (median 8 years; range 0-15 years; 89 males and 52 females) during 1995-2011. Donors were primarily unrelated and mismatched related. In the GVHD prophylaxis group, 29% and 8.6% of patients developed grade II-IV and III-IV GVHD, respectively. Of the 32 evaluable patients, 16% developed chronic [limited (n = 4) and extensive (n = 1)] GVHD. In the acute GVHD treatment group, 61% had decreased grade. In the chronic GVHD treatment group, 36% had improved symptoms. Combined immunosuppressant was reduced or discontinued in 61% patients. Major adverse events (AEs) were neutropenia (4.3%), infection (3.5%), thrombocytopenia (2.1%), myelosuppression (2.1%), and diarrhea (1.4%). MMF dosage was reduced in two children due to grade ≥ 3 AEs; two children died from infection. MMF thus may be well tolerated in children, and may be an effective option for prophylaxis and treatment of acute and chronic GVHD.

Background: Patients with relapsed or refractory neuroblastoma have a poor prognosis; there are limited effective and safe rescue chemotherapies for these patients. Development of new chemotherapy regimens for these patients is a key imperative. Procedure: We retrospectively analyzed patients with refractory or relapsed neuroblastoma who received irinotecan, etoposide, and carboplatin (IREC) as a second-line treatment for neuroblastoma. We evaluated the therapeutic response, toxicity, and survival outcomes. We also assessed the impact of UGT1A1 gene polymorphisms, which are involved in irinotecan metabolism, on the outcomes and toxicity. Results: A total of 131 cycles of IREC were administered to 43 patients with a median of two cycles per patient (range, 1–10). All patients were classified as high-risk (International Neuroblastoma Risk Group). Seven patients had relapsed before IREC. One patient (2%) showed partial response and 37 patients (86%) developed stable disease (disease control rate: 88%). Grade IV neutropenia was observed in 127 cycles (97%), while ≥ grade III gastrointestinal toxicity was observed in 3 cycles (2%). There was no IREC-related mortality. The one-year overall survival and progression-free survival rates were 65% and 52%, respectively. Patients with UGT1A1 polymorphisms showed a higher frequency of grade IV neutropenia; however, there was no increase in treatment-related mortality or nonhematological toxicity in these patients. Patients with UGT1A1 gene polymorphisms showed better one-year survival rate than the wild type (80% vs. 44%, p = 0.012). Conclusions: This study suggests that IREC is well-tolerated by patients with UGT1A1 polymorphisms and is a promising second-line chemotherapy for refractory/relapsed neuroblastoma.

Background: Central venous catheters (CVCs) have been essential devices for the treatment of children with hematological and oncological disorders. Only few studies investigated the complications and selections of different types of CVCs in these pediatric patients. This study aimed to compare risk factors for unplanned removal of two commonly used CVCs, i.e., peripherally inserted central catheters (PICCs) and tunneled CVCs, and propose better device selection for the patient. Procedure: This retrospective, single center cohort analysis was conducted on pediatric patients with hematological and oncological disorders inserted with either a PICC or a tunneled CVC. Results: Between January 1, 2013, and December 31, 2015, 89 patients inserted with tunneled CVCs (total 21,395 catheter-days) and 84 with PICCs (total 9,177 catheter-days) were followed up until the catheter removal. The median duration of catheterization was 88 days in PICCs and 186 days in tunneled CVCs (p = 1.24×10-9). PICCs at the 3-month cumulative incidence of catheter occlusion (5.2% vs. 0%, p = 4.08×10-3) and total unplanned removal (29.0% vs 7.0%, p = 0.0316) were significantly higher, whereas no significant difference was observed in the cumulative incidence of central line-associated bloodstream infection (11.8% vs. 2.3%, p = 0.664). Multivariable analysis identified younger age (

In patients with acquired AA, PGF is a major cause of cytopenia after hematopoietic stem cell transplantation. An increased incidence of PGF, especially sPGF, has been noted after the introduction of the FLU/CY regimen in children with acquired AA. To clarify the risk factors for sPGF, the clinical data of 49 patients (median age, 11 years; range, 1-19 years) with AA who received allogeneic BMT at Nagoya University Hospital from 1997 to 2016 were analyzed. Out of the 49 patients, 7 developed sPGF, and the 5-year CI was 0.15 (95% CI, 0.04-0.25). Five received the FLU/CY regimen, and the 5-year CI of sPGF was significantly higher in patients who received the regimen (0.36; 95% CI, 0.12-0.62) than in those who were conditioned with the non-FLU/CY regimen (0.06; 95% CI, 0.01-0.17; P = .01). The multivariate analysis confirmed that the FLU/CY regimen (hazard ratio, 6.12; 95% CI, 1.16-32.4; P = .03) was a significant risk factor for sPGF. sPGF improved spontaneously without stem cell boost infusions in 5 patients, ranging from 460 to 3539 days after BMT. The 10-year CI of the spontaneous trilineage recovery was 0.83 (95% CI, 0.00-0.97), and all 7 patients are alive. The FLU/CY regimen was identified as a risk factor for the sPGF development in patients with AA. The establishment of the optimal conditioning regimens for children with AA is warranted.

Insertional mutagenesis is an important risk with all genetically modified cell therapies, including chimeric antigen receptor (CAR)-T cell therapy used for hematological malignancies. Here we describe a new tagmentation-assisted PCR (tag-PCR) system that can determine the integration sites of transgenes without using restriction enzyme digestion (which can potentially bias the detection) and allows library preparation in fewer steps than with other methods. Using this system, we compared the integration sites of CD19-specific CAR genes in final T cell products generated by retrovirus-based and lentivirus-based gene transfer and by the piggyBac transposon system. The piggyBac system demonstrated lower preference than the retroviral system for integration near transcriptional start sites and CpG islands and higher preference than the lentiviral system for integration into genomic safe harbors. Integration into or near proto-oncogenes was similar in all three systems. Tag-PCR mapping is a useful technique for assessing the risk of insertional mutagenesis., Highlights • We developed a new tagmentation-assisted PCR technique (tag-PCR) to map the integration sites of genetically modified cells. • Tag-PCR enables a fast, simple, and comprehensive integration site mapping of CD19 CAR-T cells. • The insertional mutagenesis risk of piggyBac CAR-T cells appeared to be comparable to that of retroviral vector systems.

Congenital dyserythropoietic anemia (CDA) is a heterogeneous group of rare congenital disorders characterized by ineffective erythropoiesis and dysplastic changes in erythroblasts. Diagnosis of CDA is based primarily on the morphology of bone marrow erythroblasts; however, genetic tests have recently become more important. Here, we performed genetic analysis of 10 Japanese patients who had been diagnosed with CDA based on laboratory findings and morphological characteristics. We examined 10 CDA patients via central review of bone marrow morphology and genetic analysis for congenital bone marrow failure syndromes. Sanger sequencing for CDAN1, SEC23B, and KLF1 was performed for all patients. We performed whole-exome sequencing in patients without mutation in these genes. Three patients carried pathogenic CDAN1 mutations, whereas no SEC23B mutations were identified in our cohort. WES unexpectedly identified gene mutations known to cause congenital hemolytic anemia in two patients: canonical G6PD p.Val394Leu mutation and SPTA1 p.Arg28His mutation. Comprehensive genetic analysis is warranted for more effective diagnosis of patients with suspected CDA.

Introduction: Chimeric antigen receptor-modified T cells targeting CD19 (CD19.CAR-T cells) have shown clinical success in patients with hematological malignancies. Despite the encouraging results obtained with this novel therapy, a major concern to its global spread, particularly in developing countries, is its high cost. We developed a method of non-viral gene transfer using piggyBac transposon to reduce the cost of CAR-T therapy. In preclinical study, the median number and transduction efficiency of CAR-T cells obtained from 2x10 7 PBMC in 9 donors were 1.0x10 8 (range, 0.58-1.8x10 8) and 51% (range, 29-73%), respectively. The major subset of CAR-T cells was phenotypically CD8+CD45RA+CCR7+, closely related T-memory stem cells. Ex vivo, CD19.CAR-T cells showed cytotoxic effect on CD19 positive tumor cell lines. In NSG mice model, CD19.CAR-T cells successfully inhibit tumor growth. CAR gene integration sites were determined by inverse polymerase chain reaction and subsequent next-generation sequencing using MiSeq and equally distributed throughout the genome without preference for specific sites. The pre-clinical testing in mouse demonstrated safe toxicity profile at the 50 times dose of CD19.CAR-T cells. We started a human clinical trial to define feasibility, toxicity, maximum tolerated dose and clinical response of CD19.CAR-T cells (jRCTa040190099). Methods: We report the results of cohort 1 of the study in which the safety and efficacy of autologous CD19.CAR-T in patients with relapsed or refractory B-precursor acute lymphoblastic leukemia were evaluated. We engineered autologous T cells via the piggyBac transposon system with CD19.CAR-expression transposon vector and piggyBac transposase-expression vector to express CD19.CAR incorporating CD28 costimulatory domain. We designed this phase I trial using a modified 3 + 3 design to enroll 3-12 patients with relapsed or refractory acute lymphoblastic leukemia in both children and adults. In this study, patients in cohorts 1 (16-60 years old) and 2 (1-15 years old) receive 1 × 10 5 CAR-transduced T cells per kg. Patients in cohorts 3 and 4 (both 1-60 years old) receive 3 × 10 5 and 1 × 10 6 CAR-transduced T cells per kg, respectively. All patients receive 25mg/m 2/d of fludarabine and 250mg/m 2/d of cyclophosphamide for 3 days followed by a single infusion of CAR-T cells. Results: Three patients were enrolled in cohort 1 and infused with 1 × 10 5 CAR-transduced T cells per kg. All patients had previously undergone allogeneic hematopoietic stem cell transplantation. All patients had achieved a hematological complete response with salvage treatment before CAR-T therapy. None of the patients had dose-limiting toxicities (DLT) defined as nonhematological toxicities above grade 4 or cytokine release syndrome (CRS) above grade 4 or graft versus host disease (GVHD) above 4, or grade 3 nonhematological toxicities and GVHD not improved to grade 2 within 4 weeks after CAR-T infusion. There was no occurrence of non-hematological adverse events above grade 3. CRS was observed in one patient (grade 1) who also developed headache due to infiltration of CAR-T cells into the spinal fluid. In two patients, B cell aplasia lasted 2 and 9 months, respectively. Elevation of serum cytokine levels was observed in all patients and the peak time point was 7-21 days after CAR-T cell infusion. Conclusions: CD19.CAR-T cell infusion produced by the piggyBac transposon gene engineering system was safe in cohort 1 of our study. As no patients had DLT in cohort 1, we are enrolling the patients in further cohorts. Disclosures Murata: MSD: Honoraria; Kyowa Kirin: Honoraria; Sumitomo Dainippon Pharma: Honoraria; FUJIFILM: Honoraria; Toyama Chemical: Honoraria; Novartis: Honoraria; JCR Pharmaceutical: Honoraria; Astellas: Honoraria; Miyarisan Pharmaceutical: Honoraria; Asahi Kasei: Honoraria; GlaxoSmithKline: Honoraria; Celgene: Honoraria; Otsuka Pharmaceutical: Honoraria.

Introduction Head hair is a symbol of vitality, and hair loss by alopecia areata (AA) presents a burden on patients. Although traditional Japanese Kampo medicine (JKM) formulas, acupuncture, and moxibustion have historically been used for treating AA, no studies have utilized a combination of these modalities. Patient concerns A 34-year-old male with a history of childhood asthma presented with a sudden hair loss at the top of his head without any preceding symptoms. Except for a hairless patch of 5 cm × 6 cm, his general appearance was otherwise good. The patient underwent topical immunotherapy on visiting a dermatologist. However, the patient noticed an exacerbation of his hairless lesion. Diagnosis The AA diagnosis was established based on clinical appearance and dermatological findings. The Severity of Alopecia Tool (SALT) score for alopecia was 19% at diagnosis. Interventions The patient received 2 JKM formulas (saikokaryukotsuboreito and shichimotsukokato) in combination with acupuncture. When relapse occurred, a novel self-administration of pine-needle acupuncture was initiated in combination with the JKM formulas. Outcomes A 50% recovery from the baseline SALT score was achieved using JKM formulas in combination with acupuncture for 4 months. The patient achieved complete remission for 5 months. However, another stressful event induced an AA relapse with multiple lesions harboring a SALT score of 13%. Pine-needle acupuncture was initiated, resulting in faster resolution than the first treatment. Recoveries of 50% and 75% were achieved 3 and 4 months after relapse, respectively, and a long-lasting response without relapse was obtained for at least 3 years. Conclusion A combination of multimodal traditional therapies, including JKM formulas, acupuncture, and self-administered pine-needle stimulation, represents an effective integrative treatment for patients with AA.

BACKGROUND: Inherited bone marrow failure syndromes (IBMFSs) are a heterogeneous group of genetic disorders characterized by bone marrow failure, physical anomalies, and various kinds of organ complications. In addition to classical IBMFSs, such as Fanconi anemia, Diamond-Blackfan anemia, Dyskeratosis congenita, Shwachman-Diamond syndrome, and familial platelet disorders, many types of unclassified IBMFSs are reported. Over 100 genes are considered causative genes; however, the precise genetic diagnosis of IBMFSs remains challenging. We developed a capture-based target sequencing method for IBMFSs that covers more than 180 associated genes. Our system achieved genetic diagnosis for 225 (35%) of 738 patients between 2013 and 2018. However, the causative gene remained unknown for 513 (65%) patients, and further genetic analysis of these "target-negative" cases was necessary to achieve a precise diagnosis. METHODS: We performed whole exome sequencing (WES) for patients who were "target-negative" but strongly suspected of having IBMFS based on the following clinical characteristics: physical or organ anomalies (skin, nail, hair, skeletal, growth, cardiac, lung, liver, or genitourinary), family history of hematological disorder, young age (≤2 years), short telomere length ( RESULTS: Among the 513 "target-negative" cases, 166 patients were evaluated, of whom 17 patients' parents were also analyzed in a trio-based analysis. New pathogenic variants were identified in 18 of the 166 (11%) patients according to the American College of Medical Genetics (ACMG) guidelines, of which 5 variants were revealed to be de novo. Diagnostic variants were identified in FANCF, SRP54, RPL19, RPL5, RTEL1, RUNX1, MECOM, CDC42, GNE, SLNF14 (all n = 1). In addition to IBMFS-associated genes, causative genes for congenital hemolytic anemia (G6PD, PKLR), inborn error of metabolism (SLC46A1), and primary immune deficiency (NFKB2, LRBA) are also identified (all n = 1). Moreover, loss-of-function mutation of ADH5 gene are identified in three patients that seems to be associated to novel IBMFSs. On the other hand, no pathogenic variant in GATA2, ERCC6L2, LIG4, and SAMD9/SAMD9L genes that are reported as unclassified IBMFSs in Europe and United States are identified in our cohort. CONCLUSION: Our findings support the utility of WES (especially trio-based analysis) as a diagnostic tool for IBMFSs. Furthermore, genetic background of IBMFSs in East Asia seems to be different from that of Europe and United States. Disclosures No relevant conflicts of interest to declare.

Background Viral infection is one of the major causes of mortality in patients undergoing hematopoietic stem cell transplant (HSCT). Systemic infection of adenovirus (AdV) has emerged as a not uncommon viral infection with significant morbidity and mortality as with cytomegalovirus and Epstein–Barr virus infection. Routine surveillance for these viruses has become a clinical practice and subsequent preemptive therapy improves patients' outcomes; however, the effectiveness of preemptive therapy for AdV has not been fully investigated in patients with a lethal form of AdV infection. Methods Sequential AdV loads were retrospectively analyzed in children with fulminant AdV hepatitis after HSCT. Results The AdV DNA became detectable (1 × 104 copies/mL) as early as 2 weeks after HSCT. These levels reached >1 × 108 copies/mL at the onset of fulminant hepatitis. However, we determined that γ-glutamyltransferase levels were elevated to >100 IU/L at least 2 weeks before the diagnosis of hepatitis. Conclusions Our observation raises the possibility that elevated γ-glutamyltransferase could be a sentinel marker for AdV hepatitis, which prompts elaborated monitoring of AdV load and targeted treatment.

Background Distinguishing hypocellular refractory cytopenia of childhood (RCC) from aplastic anemia (AA) is challenging. Thus far, no studies have compared the cytokine profiles in patients with AA to those with hypocellular RCC. In the present study, we addressed whether thrombopoietin (TPO) and interleukin 17 (IL-17) plasma levels are useful for differentiating between the two diseases. Methods We measured the endogenous plasma concentrations of TPO and IL-17 in 29 patients with AA, 34 patients with hypocellular RCC, and 31 healthy controls using sensitive enzyme-linked immunosorbent assays. Results The TPO and IL-17 plasma levels were significantly lower in patients with hypocellular RCC than in patients with AA (P < 0.001 and P = 0.007, respectively). The multivariate logistic regression analysis identified moderate disease severity, TPO levels of

Background Juvenile myelomonocytic leukemia (JMML) is a rare and exclusively pediatric myelodysplastic/myeloproliferative neoplasm. This disease is genetically characterized by an extremely small number of somatic mutations (an average of 0.8 mutations/exome/patient). It has been shown that causative somatic and/or germline mutations activating the RAS pathway are located in PTPN11, NF1, NRAS, KRAS, and CBL in 85% of patients with JMML. Furthermore, up to 20% of the patients have additional secondary mutations including SETBP1, and JAK3 mutations. In 2% of the patients, we identified, by RNA sequencing, activating kinase lesions affecting ALK or ROS1. Such findings suggest that other kinase fusions are present in JMML. There is an exceptional scarcity of somatic passenger mutations on the exome, suggesting that a small number of driver mutations drive JMML. However, to date, this hypothesis has not been investigated by whole-genome sequencing. Patients and Methods We performed a whole-genome sequencing (WGS) study in 48 patients with JMML. Bone marrow specimens and in vitro-cultured T cells were used as tumor and germline samples, respectively. Next-generation sequencing was performed using a HiSeq X platform (Illumina). Data analysis was performed by our in-house pipeline. Specifically, the pipeline detects single nucleotide variants (SNVs), copy number variants, somatic loss of heterozygosity (LOH), and chromosomal structural variations (SVs). The study was approved by the institutional review board of Nagoya University Graduate School of Medicine. Results In each patient we detected an average of 28 somatic mutations. These were primarily C-to-T transition in the CpG context, indicating that the mutations occurred by cell division. Besides RAS pathway and known secondary mutations, we observed no significant accumulation of somatic mutations in either coding or non-coding regions. Although we detected RAS pathway mutations in 90% of the patients, all mutations were on exome. However, we identified germline microdeletions affecting CBL and NF1, which had not been identified by exome sequencing. Additionally, we found two LOH events that affected NF1. Bi-allelic inactivation of NF1 is generally observed in patients with JMML; however, no pathogenic SNVs were identified in these two patients. We identified two chromosomal translocations that caused activating kinase lesions (i.e., RANBP2-ALK and TBL1XR1-ROS1). These had been pointed out in our previous RNA sequencing study. Another patient carried a complex SV that affected XPO1 (encoding exportin 1 or chromosome region maintenance 1 protein homolog). Although fusion genes involving XPO1 are reported to be present in lymphoid malignancies, the role of this SV in JMML remains unclear. Conclusions JMML is characterized by driver mutations that are largely present within the exome. However, WGS can still play a role in identifying both coding and non-coding mutations. LOH events without pathogenic SNVs suggest the presence of novel regulatory mechanisms of NF1. Conclusively, JMML is characterized by a paucity of somatic alterations and driver mutations. Hence, current research efforts should focus on RAS pathway mutations and known secondary mutations, many of which can be targeted. Disclosures No relevant conflicts of interest to declare.

Introduction: Aplastic anemia (AA) is an autoimmune-mediated disease with a complex mechanism. Innate as well as acquired immune system have been considered to participate in its pathogenesis. Although several studies have demonstrated the role of T and B cells in AA, there is limited research focusing on natural killer (NK) cells, which are important in innate and adaptive immune system. NK cells interact with T cells and alter their immune responses in a variety of diseases. Recent investigation suggests that NK cells serve immunoregulatory roles by attenuating autologous CD8+ T-cell response in AA (Chen et al. Cell Immunol 335:6-14, 2019). Here we assessed the kinetics of NK cells before and after immunosuppressive therapy (IST) in patients with AA to determine the association between NK cells and clinical courses. Methods: Patients with severe AA, in Japan, who required IST as first-line therapy were prospectively enrolled between May 2012 and October 2017. The IST regimen comprised rabbit anti-thymocyte globulin (ATG, thymoglobulin®, 2.5 or 3.5 mg/kg/day for 5 days), cyclosporine A (6 mg/kg/day for minimum 6 months), and methylprednisolone (2 mg/kg/day for 5 days) with subsequent tapering of the dose for 28 days. Flow cytometry was used to assess lymphocyte subsets, consisting of CD3+, CD4+, and CD8+ T cells, CD19+ B cells, CD56+ NK cells, and CD4+CD25+CD127+ regulatory T cells (Tregs), before and after ATG administration on days 0, 14, 28, 60, 90, 120, and 180. Plasma rabbit ATG levels were measured using a rabbit IgG ELISA kit on days 14 and 28 (Bethyl Laboratories, Montgomery, TX, USA). Receiver-operator characteristic (ROC) curves were generated to differentiate between response and no response to IST. Results: A total of 81 patients (aged 1.7-67.9 years) were randomized; 43 and 38 patients received 2.5 and 3.5 mg/kg of r-ATG, respectively. Median follow-up duration was 445 days (range; 183-2165 days) from first ATG infusion. After 6 months, 50 patients (58%) responded to the therapy, including 4 (5%) who achieved a complete response and 45 (56%) a partial response. The response rates did not differ significantly between the two dosages in ATG groups at 6 months (2.5 mg/kg, 63% vs. 3.5 mg/kg, 58%, P = 0.820). The median absolute number of NK cells before IST was 92/µL (0-1,085/µL). To judge patients' responses to IST, we defined 138/µL as the absolute number of NK cells count before IST as the cut-off value using ROC curves. The response rate of patients with higher NK cells (≥ 138 /mL) to IST at 6 months was significantly higher than those with lower NK cells (Figure 1, < 138 /mL, 78% vs. 52%, P = 0.031). Multivariate logistic regression analysis identified the absolute number of NK cells before IST and reticulocyte at diagnosis (odds ratio [OR] = 0.16; 95% confidence interval [CI], 0.04-0.62; P = 0.008 and OR = 0.20; 95% CI, 0.06-0.67; P = 0.009, respectively) and the r-ATG plasma concentration on day 28 (OR = 0.11; 95% CI, 0.03-0.39; P = 0.001) as independent predictors of response to IST. The median absolute number of NK cells on days 90, 120, and 180 after IST was higher in the responders (113/mL, 118/mL, and 159/mL, respectively) than in the non-responders (57/mL, 53/mL, and 64/mL, P = 0.001, P Conclusions: The current study indicates that NK cells play a role in the pathogenesis of AA and may be the predictors of response to IST. Recovery of NK cell count correlated with hematopoietic recovery following IST, indicating that they may be used as markers to assess disease activity, treatment response and relapse. Disclosures Yamazaki: Novartis Pharma K.K.: Honoraria; Sanofi K.K.: Honoraria; Nippon Shinyaku Co., Ltd.: Honoraria. Nakao:Ono Pharmaceutical: Honoraria; Kyowa Kirin: Honoraria; Alaxion Pharmaceuticals: Honoraria; Ohtsuka Pharmaceutical: Honoraria; Celgene: Honoraria; Daiichi-Sankyo Company, Limited: Honoraria; Janssen Pharmaceutical K.K.: Honoraria; SynBio Pharmaceuticals: Consultancy; Novartis Pharma K.K: Honoraria; Bristol-Myers Squibb: Honoraria; Takeda Pharmaceutical Company Limited: Honoraria; Chugai Pharmaceutical Co.,Ltd: Honoraria.

Background Juvenile myelomonocytic leukemia (JMML) is a rare myelodysplastic/ myeloproliferative neoplasm that occurs during infancy and early childhood. The clinical course of the disease varies widely. The majority of children require allogenic hematopoietic stem cell transplantation (HSCT) for long term survival, but the disease will eventually resolve spontaneously in ~15% of patients. Previous studies have identified clinical and molecular risk factors in JMML. More recently, three groups independently discovered that genome-wide methylation profiling using 450K Illumina array revealed that the high methylation (HM) subgroup was significantly associated with poor survival compared to the low methylation (LM) subgroup (Murakami 2018 Blood, Stieglitz 2017 Nat. Commun., Lipka 2017 Nat. Commun.). 450K could be a standard assay for stratification of JMML. However, it is now unavailable because the manufacture replaced it with EPIC array. Here, we developed a next-generation sequencing-based clinical test recapitulate 450K clustering results using the digital restriction enzyme analysis of methylation (DREAM) method (Jelinek 2012 Epigenetics). Patients and Methods We studied 99 children (67 boys and 32 girls) with JMML. All the patients were included in our previous publications. First, we assessed JMML samples with DREAM. Briefly, genomic DNA was sequentially cut with two enzymes SmaI and XmaI recognizing the same sequence, CCCGGG sites in DNA. Enzyme-treated DNA was then used to generate sequencing libraries according to the Illumina protocols, and run on an Illumina Hiseq 2500. We assessed 10 JMML samples with reduced representation bisulfite sequencing (RRBS) (Meissner 2005 Nucleic Acids Res.). In brief, purified genomic DNA was digested by the methylation-insensitive restriction enzyme MspI to generate short fragments that contain CpG dinucleotides at the ends. The CpG-rich DNA fragments (40-220 bp) were size selected, subjected to bisulfite conversion, PCR amplified and end sequenced on an Illumina Genome analyzer. Results We analyzed 99 samples using the DREAM with 8.87 (4.09-16.35) million reads (median, [range]), and determined methylation level in 62,525 (52,356-75,185) CpG sites (median [range]). We observed a strong correlation between DREAM methylation ratio and 450K beta-value of overlapping CpG sites (Pearson r2 = 0.95 [0.913-0.962], median [range]). We performed unsupervised consensus clustering with DREAM methylation data of 7,704 CpG sites within ±1 kb from TSS on autosomal chromosomes detected in ≥95% of the samples with imputation of the missing data using the median of each CpG site methylation level. Clustering identified two distinct subgroups, the HM subgroup (n = 35) and the LM subgroup (n = 64), matching 95% (94 of 99) with the 450K clustering results. The HM subgroup patients showed significantly poorer 5-year OS than the LM subgroup patients (41.9% [95% confidence interval {CI}], 25.3%-57.6%) vs. 71.4% [95% CI, 56.2%-82.1%]; P = 0.00345). Discrepancies in the clustering results between DREAM and 450K were observed in only 5 patients (2 survived and 3 died); all 5 patients were reclassified as those with LM with DREAM from being HM with 450K. We also performed RRBS methylation analysis on 10 patients. Unsupervised consensus clustering using promoter-associated 4,971 CpG sites measured with RRBS identified HM (n = 5) and LM (n = 5) subgroups and completely matched with the classification made using DREAM and 450K. Then, we developed a prediction model of the methylation subgroups using a machine-learning program. We selected 85 CpG sites from 7,704 CpG sites used for unsupervised clustering of the DREAM assay that showed a distinct difference in the average methylation level (>0.3) between the HM and LM subgroups of the learning cohort (n = 70) and developed a support vector machine (SVM) model. As a validation cohort, we analyzed the remaining 29 JMML samples with a SVM model and confirmed a high matching rate with 450K clustering results (100%, 29 of 29). Conclusions We could develop a methylation test for JMML using the DREAM assay. Both the unsupervised clustering analysis and SVM model could repeat the result of 450K-based methylation classification, i.e., the HM and LM subgroups. The relatively lower cost of the DREAM assay (US$200/sample) enabled us to incorporate methylation classification in JMML in most settings. Disclosures No relevant conflicts of interest to declare.

Therapy for BK virus (BKV)–associated hemorrhagic cystitis (BKV-HC) is limited after hematopoietic stem cell transplantation (HSCT). We examined whether choreito, a formula from Japanese traditional Kampo medicine, is effective for treating BKV-HC. Among children who underwent allogeneic HSCT between October 2006 and March 2014, 14 were diagnosed with BKV-HC (median, 36 days; range, 14 to 330 days) after HSCT, and 6 consecutive children received pharmaceutical-grade choreito extract granules. The hematuria grade before treatment was significantly higher in the choreito group than in the nonchoreito group (P = .018). The duration from therapy to complete resolution was significantly shorter in the choreito group (median, 9 days; range, 4 to 17 days) than in the nonchoreito group (median, 17 days; range, 15 to 66 days; P = .037). In 11 children with macroscopic hematuria, the duration from treatment to resolution of macroscopic hematuria was significantly shorter in the choreito group than in the nonchoreito group (median, 2 days versus 11 days; P = .0043). The BKV load in urine was significantly decreased 1 month after choreito administration. No adverse effects related to choreito administration were observed. Choreito may be a safe and considerably promising therapy for the hemostasis of BKV-HC after HSCT.

Juvenile myelomonocytic leukemia (JMML), a rare and aggressive myelodysplastic/myeloproliferative neoplasm that occurs in infants and during early childhood, is characterized by excessive myelomonocytic cell proliferation. More than 80% of patients harbor germ line and somatic mutations in RAS pathway genes (eg, PTPN11, NF1, NRAS, KRAS, and CBL), and previous studies have identified several biomarkers associated with poor prognosis. However, the molecular pathogenesis of 10% to 20% of patients and the relationships among these biomarkers have not been well defined. To address these issues, we performed an integrated molecular analysis of samples from 150 JMML patients. RNA-sequencing identified ALK/ROS1 tyrosine kinase fusions (DCTN1-ALK, RANBP2-ALK, and TBL1XR1-ROS1) in 3 of 16 patients (18%) who lacked canonical RAS pathway mutations. Crizotinib, an ALK/ROS1 inhibitor, markedly suppressed ALK/ROS1 fusion-positive JMML cell proliferation in vitro. Therefore, we administered crizotinib to a chemotherapy-resistant patient with the RANBP2-ALK fusion who subsequently achieved complete molecular remission. In addition, crizotinib also suppressed proliferation of JMML cells with canonical RAS pathway mutations. Genome-wide methylation analysis identified a hypermethylation profile resembling that of acute myeloid leukemia (AML), which correlated significantly with genetic markers with poor outcomes such as PTPN11/NF1 gene mutations, 2 or more genetic mutations, an AML-type expression profile, and LIN28B expression. In summary, we identified recurrent activated ALK/ROS1 fusions in JMML patients without canonical RAS pathway gene mutations and revealed the relationships among biomarkers for JMML. Crizotinib is a promising candidate drug for the treatment of JMML, particularly in patients with ALK/ROS1 fusions.

Acute immune thrombocytopenia (ITP) is a common benign bleeding disorder of variable etiology characterized by isolated thrombocytopenia. Intravenous immunoglobulin (IVIG) treatment is generally given as an initial treatment to pediatric patients with ITP, but markers predictive of the response to IVIG remain poorly defined. We retrospectively evaluated whether clinical and laboratory findings before treatment could predict response to IVIG and progression to chronic ITP in Japanese children with ITP. Between April 1997 and December 2011, a total of 49 children with newly diagnosed ITP were initially treated with IVIG. Their medical records were retrospectively reviewed. In multivariate analyses, lower white blood cell (WBC) count was the only unfavorable factor for response to IVIG and progression to chronic ITP. Patients with WBC count7.0 × 10(9)/L had a lower probability of thrombocytopenia-free survival (41 vs. 77 %, P = 0.003) and a higher rate of progression to chronic ITP (29 vs. 6 %, P = 0.040) than those with WBC count ≥7.0 × 10(9)/L. These results suggest that ITP with lower WBC count may represent a distinct subgroup requiring initial treatment other than IVIG.

Background In the treatment of bifurcation lesions, routine stenting of both branches has thus far failed to demonstrate a clear clinical advantage over a provisional one-stent strategy. On the other hand, large scale data evaluating different stent types for clinical outcomes after one-stent treatment with final kissing inflation (FKI) of bifurcation lesions is also limited. This prospective study evaluated the clinical and angiographic outcomes of paclitaxel-eluting stents (PES) vs. sirolimus-eluting stents (SES) in single crossover main branch stenting followed by FKI in patients with bifurcation lesions. Methods We randomized 800 patients with single bifurcation lesions to PES (n=400) and SES (n=400) groups. Results Crossover rates to the two-stent strategy were low in both groups (PES 1.5%, SES 2.8%; p=0.23). At 1year, there was no significant difference in the primary endpoint of this study, target lesion revascularization rate (PES 3.8%, SES 3.2%, hazard ratio 0.83; 95% confidence interval 0.39 to 1.76; p=0.62). Stent thrombosis occurred in only 1 case in the SES group after 282days. At 9months, a total of 593 patients underwent quantitative coronary measurement. The main branch restenosis rate in the PES group was significantly higher than that of the SES group (PES 12.2%, SES 5.5%; p=0.004), however both groups exhibited similar high side branch restenosis rates (PES 17.2%, SES 19.3%; p=0.6). Conclusions In patients with bifurcation lesions, a single stent strategy using PES and SES with FKI indicated similar 1year clinical outcomes and safety profiles.

Pulmonary hypertension (PH) is an infrequently reported complication after hematopoietic stem cell transplantation, and its etiology and therapeutic strategies, especially in infants, remain unclear. We report a case of severe PH that developed in an infant with acute leukemia following administration of busulfan as a preconditioner for cord blood transplantation; the case was successfully treated with sildenafil and beraprost, which to our knowledge is the first reported successful use of this regimen in PH following transplantation for infantile leukemia. From a review of all previous reports, use of busulfan in infants may raise the risk of developing PH, and unlike definitive pulmonary veno-occlusive disease, PH in this subgroup may be reversible by early detection and treatment.