Scoring system for diagnosis and pretreatment risk assessment of neuroblastoma using urinary biomarker combinations.

The urinary catecholamine metabolites, homovanillic acid (HVA) and vanillylmandelic acid (VMA), are used for the adjunctive diagnosis of neuroblastomas. We aimed to develop a scoring system for the diagnosis and pretreatment risk assessment of neuroblastoma, incorporating age and other urinary catecholamine metabolite combinations. Urine samples from 227 controls (227 samples) and 68 patients with neuroblastoma (228 samples) were evaluated. First, the catecholamine metabolites vanillactic acid (VLA) and 3-methoxytyramine sulfate (MTS) were identified as urinary marker candidates through comprehensive analysis using liquid chromatography-mass spectrometry. The concentrations of these marker candidates and conventional markers were then compared among controls, patients, and numerous risk groups to develop a scoring system. Participants were classified into four groups: control, low risk, intermediate risk, and high risk, and the proportional odds model was fitted using the L2-penalized maximum likelihood method, incorporating age on a monthly scale for adjustment. This scoring model using the novel urine catecholamine metabolite combinations, VLA and MTS, had greater area under the curve values than the model using HVA and VMA for diagnosis (0.978 vs. 0.964), pretreatment risk assessment (low and intermediate risk vs. high risk: 0.866 vs. 0.724; low risk vs. intermediate and high risk: 0.871 vs. 0.680), and prognostic factors (MYCN status: 0.741 vs. 0.369, histology: 0.932 vs. 0.747). The new system also had greater accuracy in detecting missing high-risk neuroblastomas, and in predicting the pretreatment risk at the time of screening. The new scoring system employing VLA and MTS has the potential to replace the conventional adjunctive diagnostic method using HVA and VMA.

A Japanese retrospective study of non-tuberculous mycobacterial infection in children, adolescents, and young adult patients with hematologic-oncologic diseases.

Non-tuberculous mycobacterial infection (NTM) is rare in healthy children, with lymphadenitis being the most common presentation. Immunocompromised populations are known to be at high risk, but the clinical picture of NTM infection in pediatric hematology/oncology patients is unclear. In this nationwide retrospective analysis of patients under the age of 40 treated in Japanese pediatric hematology/oncology departments who developed NTM infection between January 2010 and December 2020, 36 patients (21 patients with hematopoietic stem cell transplantation (HSCT) and 15 nontransplant patients) were identified. Post-transplant patients were infected with NTM at 24 sites, including the lungs (n = 12), skin and soft tissues (n = 6), bloodstream (n = 4), and others (n = 2). Nine of twelve patients with pulmonary NTM infection had a history of pulmonary graft-versus-host disease (GVHD), and rapid-growing mycobacteria (RGM) were isolated from five of them. In nontransplant patients, the primary diseases were acute lymphoblastic leukemia (ALL; n = 5), inborn errors of immunity (IEI; n = 6), and others (n = 4). All cases of ALL had bloodstream infections with RGM, whereas all cases of IEI were infected with slow-growing mycobacteria (SGM). In summary, three typical clinical scenarios for pediatric hematology/oncology patients have been established: RGM-induced pulmonary disease in patients with pulmonary GVHD, RGM bloodstream infection in patients with ALL, and SGM infection in patients with IEI. Our findings suggest that NTM must be regarded as a pathogen for infections in these high-risk patients, especially those with pulmonary GVHD, who may require active screening for NTM.

Novel xeno-free and serum-free culturing condition to improve piggyBac transposon-based CD19 chimeric antigen receptor T-cell production and characteristics.

BACKGROUND AIMS: Chimeric antigen receptor (CAR) T cell is a novel therapy for relapse and refractory hematologic malignancy. Characteristics of CAR T cells are associated with clinical efficacy and toxicity. The type of serum supplements used during cultivation affects the immunophenotype and function of viral-based CAR T cells. This study explores the effect of serum supplements on nonviral piggyBac transposon CAR T-cell production. METHODS: PiggyBac CD19 CAR T cells were expanded in cultured conditions containing fetal bovine serum, human AB serum or xeno-free serum replacement. We evaluated the effect of different serum supplements on cell expansion, transduction efficiency, immunophenotypes and antitumor activity. RESULTS: Xeno-free serum replacement exhibited comparable CAR surface expression, cell expansion and short-term antitumor activity compared with conventional serum supplements. However, CAR T cells cultivated with xeno-free serum replacement exhibited an increased naïve/stem cell memory population and better T-cell expansion after long-term co-culture as well as during the tumor rechallenge assay. CONCLUSIONS: Our study supports the usage of xeno-free serum replacement as an alternative source of serum supplements for piggyBac-based CAR T-cell expansion.

[Chimeric antigen receptor (CAR) T cells targeting CD19 for acute lymphoblastic leukemia].

Chimeric antigen receptor transgenic T cell (CAR-T) therapy targeting the CD19 antigen was approved for relapsed/refractory acute lymphocytic leukemia in the United States in 2017 and in Japan in 2019. Despite the excellent efficacy of CAR-T therapy, the relapse rate is about 50%. To reduce this rate, it will be important to examine predictive factors for relapse and which patients should receive hematopoietic cell transplantation. In addition, as the high cost of CAR-T cells has become a financial toxicity that threatens the health insurance system in many countries, development of less expensive CAR-T products using non-viral vectors is also underway.

Successful treatment of a novel type I interferonopathy due to a de novo PSMB9 gene mutation with a Janus kinase inhibitor.

BACKGROUND: Type I interferonopathies are a recently established subgroup of autoinflammatory diseases caused by mutations in genes associated with proteasome degradation or cytoplasmic RNA- and DNA-sensing pathways. OBJECTIVE: This study aimed to unveil the molecular pathogenesis of a patient with novel type I interferonopathy, for which no known genetic mutations have been identified. METHODS: We performed the whole-exome sequencing of a 1-month-old boy with novel type I interferonopathy. We also investigated proteasome activities using patient-derived B lymphoblastoid cell lines (LCLs) and normal LCLs transduced with the mutant gene. RESULTS: Whole-exome sequencing identified a de novo proteasome 20S subunit beta 9 (PSMB9) p.G156D mutation in the patient who developed fever, a chilblain-like skin rash, myositis, and severe pulmonary hypertension due to the hyperactivation of IFN-α. Patient-derived LCLs revealed reduced proteasome activities, and exogenous transduction of mutant PSMB9 p.G156D into normal LCLs significantly suppressed proteasome activities, and the endogenous PSMB9 protein was lost along with the reduction of other immunoproteasome subunits, PSMB8 and PSMB10 proteins. He responded to the administration of a Janus kinase inhibitor, tofacitinib, and he was successfully withdrawn from venoarterial extracorporeal membranous oxygenation. At age 7 months, he received an unrelated cord blood transplantation. At 2 years posttransplantation, he no longer required tofacitinib and experienced no disease recurrence. CONCLUSIONS: We present the case of a patient with a novel type I interferonopathy caused by a de novo PSMB9 p.G156D mutation that suppressed the wild-type PSMB9 protein expression. Janus kinase inhibitor and stem cell transplantation could be curative therapies in patients with severe interferonopathies.

Combination of tumor necrosis factor-α and epidermal growth factor induces the adrenergic-to-mesenchymal transdifferentiation in SH-SY5Y neuroblastoma cells.

Neuroblastoma, a type of cancer that is common in children, is composed of two genetically clonal but epigenetically distinct cell types: mesenchymal (MES) and adrenergic (ADRN) types, controlled by super-enhancer-associated lineage-specific transcription factor networks. Mesenchymal-type cells are more migratory, resistant to chemotherapy, and prevalent in relapse tumors. Importantly, both cell types spontaneously transdifferentiate into one another, and this interconversion can be induced by genetic manipulations. However, the mechanisms of their spontaneous transdifferentiation and extracellular factors inducing this phenomenon have not yet been elucidated. Using a unique approach involving gene set enrichment analysis, we selected six ADRN and 10 MES candidate factors, possibly inducing ADRN and MES phenotypes, respectively. Treatment with a combination of 10 MES factors clearly induced the MES gene expression profile in ADRN-type SH-SY5Y neuroblastoma cells. Considering the effects on gene expression profile, migration ability, and chemoresistance, a combination of tumor necrosis factor alpha (TNF-α) and epidermal growth factor (EGF) was sufficient to synergistically induce the ADRN-to-MES transdifferentiation in SH-SY5Y cells. In addition, human neuroblastoma cohort analysis revealed that the expression of TNF and EGF receptors was strongly associated with MES gene expression signatures, supporting their important roles in transdifferentiation in vivo. Collectively, we propose a mechanism of neuroblastoma transdifferentiation induced by extracellular growth factors, which can be controlled in clinical situations, providing a new therapeutic possibility.

Relationship between plasma rabbit anti-thymocyte globulin concentration and immunosuppressive therapy response in patients with severe aplastic anemia.

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.

Risk factors for secondary poor graft function after bone marrow transplantation in children with acquired aplastic anemia.

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.

Development of clinical paroxysmal nocturnal haemoglobinuria in children with aplastic anaemia.

The clinical significance of paroxysmal nocturnal haemoglobinuria (PNH) in children with aplastic anaemia (AA) remains unclear. We retrospectively studied 57 children with AA between 1992 and 2010. During the follow-up, five patients developed clinical PNH, in whom somatic PIGA mutations were detected by targeted sequencing. The 10-year probability of clinical PNH development was 10·2% (95% confidence interval, 3·6-20·7%). Furthermore, the detection of minor PNH clones by flow cytometry at AA diagnosis was a risk factor for the subsequent development of clinical PNH. These patients with PNH clones at AA diagnosis should undergo periodic monitoring for potential clinical PNH development.

Allogeneic hematopoietic stem cell transplantation using ATG: prophylaxis and treatment.

Graft versus host disease (GVHD) following allogeneic hematopoietic stem cell transplantation is associated with an increase in patient transplant-related morbidity and mortality and a decrease in quality of life. Adequate immune suppression is critical to prevent GVHD without increasing the risk of developing a severe infection or relapse of malignancy. Anti-T cell globulin (ATG) combined with other immunosuppressive drugs can reduce the risk of severe acute GVHD and extensive chronic GVHD without enhancing the rate of severe infection or relapse of malignancy. Future research is warranted to determine the optimum ATG dosage, as the pharmacokinetics of ATG substantially differs among patients and impact transplant outcomes. Therefore, further studies are required to determine the therapeutic potential of ATG for steroid refractory GVHD.

Regulation of adoptive immunotherapy.

Adoptive immunotherapy using chimeric antigen receptor (CAR)-modified T cells has provided a major breakthrough in the treatment of hematological malignancies. In Japan, it is expected that CD19 CAR-T cell therapy will be introduced earlier in clinical B cell malignancy settings and/or that a novel CAR-T cell therapy will be developed for non-B cell malignancies. The "Act on the Safety of Regenerative Medicine" and the "Revised Pharmaceutical Affairs Act" were promulgated in 2014. Both Acts were very important to the introduction and development of CAR-T therapy in Japan. "Specified cell products" produced according to the former Act can be used in clinical research projects, while "regenerated medical products" produced according to the latter Act can be used in clinical trials, having been launched on the market. In this chapter, we will summarize the regulations pertaining to adoptive immunotherapy on the basis of these two essential Acts.

Paroxysmal nocturnal hemoglobinuria and telomere length predicts response to immunosuppressive therapy in pediatric aplastic anemia.

Acquired aplastic anemia is an immune-mediated disease characterized by severe defects in stem cell number resulting in hypocellular marrow and peripheral blood cytopenias. Minor paroxysmal nocturnal hemoglobinuria populations and a short telomere length were identified as predictive biomarkers of immunosuppressive therapy responsiveness in aplastic anemia. We enrolled 113 aplastic anemia patients (63 boys and 50 girls) in this study to evaluate their response to immunosuppressive therapy. The paroxysmal nocturnal hemoglobinuria populations and telomere length were detected by flow cytometry. Forty-seven patients (42%) carried a minor paroxysmal nocturnal hemoglobinuria population. The median telomere length of aplastic anemia patients was -0.99 standard deviation (SD) (range -4.01-+3.01 SD). Overall, 60 patients (53%) responded to immunosuppressive therapy after six months. Multivariate logistic regression analysis identified the absence of a paroxysmal nocturnal hemoglobinuria population and a shorter telomere length as independent unfavorable predictors of immunosuppressive therapy response at six months. The cohort was stratified into a group of poor prognosis (paroxysmal nocturnal hemoglobinuria negative and shorter telomere length; 37 patients) and good prognosis (paroxysmal nocturnal hemoglobinuria positive and/or longer telomere length; 76 patients), respectively. The response rates of the poor prognosis and good prognosis groups at six months were 19% and 70%, respectively (P<0.001). The combined absence of a minor paroxysmal nocturnal hemoglobinuria population and a short telomere length is an efficient predictor of poor immunosuppressive therapy response, which should be considered while deciding treatment options: immunosuppressive therapy or first-line hematopoietic stem cell transplantation. The trial was registered in www.umin.ac.jp with number UMIN000017972.

Somatic mosaicism for oncogenic NRAS mutations in juvenile myelomonocytic leukemia.

Juvenile myelomonocytic leukemia (JMML) is a rare pediatric myeloid neoplasm characterized by excessive proliferation of myelomonocytic cells. Somatic mutations in genes involved in GM-CSF signal transduction, such as NRAS, KRAS, PTPN11, NF1, and CBL, have been identified in more than 70% of children with JMML. In the present study, we report 2 patients with somatic mosaicism for oncogenic NRAS mutations (G12D and G12S) associated with the development of JMML. The mutated allele frequencies quantified by pyrosequencing were various and ranged from 3%-50% in BM and other somatic cells (ie, buccal smear cells, hair bulbs, or nails). Both patients experienced spontaneous improvement of clinical symptoms and leukocytosis due to JMML without hematopoietic stem cell transplantation. These patients are the first reported to have somatic mosaicism for oncogenic NRAS mutations. The clinical course of these patients suggests that NRAS mosaicism may be associated with a mild disease phenotype in JMML.

Peripheral blood lymphocyte telomere length as a predictor of response to immunosuppressive therapy in childhood aplastic anemia.

Predicting the response to immunosuppressive therapy could provide useful information to help the clinician define treatment strategies for patients with aplastic anemia. In our current study, we evaluated the relationship between telomere length of lymphocytes at diagnosis and the response to immunosuppressive therapy in 64 children with aplastic anemia, using flow fluorescence in situ hybridization. Median age of patients was ten years (range 1.5-16.2 years). Severity of the disease was classified as very severe in 23, severe in 21, and moderate in 20 patients. All patients were enrolled in multicenter studies using antithymocyte globulin and cyclosporine. The response rate to immunosuppressive therapy at six months was 52% (33 of 64). The probability of 5-year failure-free survival and overall survival were 56% (95% confidence interval (CI): 41-69%) and 97% (95%CI: 87-99%), respectively. Median telomere length in responders was -0.4 standard deviation (SD) (-2.7 to +3.0 SD) and -1.5 SD (-4.0 to +1.6 (SD)) in non-responders (P<0.001). Multivariate analysis showed that telomere length shorter than -1.0 SD (hazard ratio (HR): 22.0; 95%CI: 4.19-115; P<0.001), platelet count at diagnosis less than 25×10(9)/L (HR: 13.9; 95%CI: 2.00-96.1; P=0.008), and interval from diagnosis to immunosuppressive therapy longer than 25 days (HR: 4.81; 95%CI: 1.15-20.1; P=0.031) were the significant variables for poor response to immunosuppressive therapy. Conversely to what has been found in adult patients, measurement of the telomere length of lymphocytes at diagnosis is a promising assay in predicting the response to immunosuppressive therapy in children with aplastic anemia.

Growth-related disappearance of the childhood relationship between height and blood pressure levels.

CONTEXT: Although there is a positive relationship between height and blood pressure (BP) levels in children, there are no reports regarding the association between height and BP levels in adolescents and adults. OBJECTIVE: This study examined whether there is an association between height and BP levels in Japanese adolescents. METHODS: The source population was all fifth (10 and 11-year-olds) and ninth graders (14 and 15-year-olds) who attended 11 elementary schools and five junior high schools in the Iwata area from 2002-2008. School-based screenings were conducted annually by the local government from April to June. Data obtained from health examinations were analysed, including anthropometric measurements and BP levels, for 11 780 children (98.7% of the source population). RESULTS: Height showed significant positive relationships with systolic blood pressure (SBP) and diastolic blood pressure (DBP) in childhood and in adolescent males. In contrast, the relationship between height and SBP was significantly weaker in adolescent females than in childhood and there was no significant relationship between height and DBP in adolescent females. CONCLUSION: The relationship between height and SBP was attenuated by development in females and the relationship between height and DBP disappeared.

Safety and adherence of Umezu polyphenols in the Japanese plum (Prunus mume) in a 12-week double-blind randomized placebo-controlled pilot trial to evaluate antihypertensive effects.

OBJECTIVES: Medications or lifestyle changes to prevent or improve hypertension often press considerable efforts on patients suffering from mild hypertension. Capsules including Umezu polyphenols (UP), polyphenols in Japanese plums, may help them to control their blood pressure (BP). The aim of this study is to evaluate the effectiveness of UP on BP and its safety. METHODS: A total of 15 healthy workers without antihypertensive medication who had some concerns about their BP, preferably normal-high BP or hypertension level 1, were randomized in a double-blind manner into UP ingesting and placebo groups. Each subject was instructed to take four capsules daily for 12 weeks (daily UP dose, 800 mg for the UP ingesting group; and 0 mg for the placebo group). These subjects were followed for 12 weeks, and their BP both at home and at the examination site, as well as self-perceived quality-of-life outcomes and possible side effects, was monitored during that period. Group × time interactions on BP changes were examined. RESULTS: All of the 15 subjects completed the 12-week intervention trial. The BP changes did not significantly differ between the UP ingesting and placebo groups, neither at the examination site nor at home. But during the study period, no adverse effects were observed. CONCLUSIONS: No remarkable effect of UP on BP was observed. However, a higher dose of UP was confirmed safe and high in adherence in this 12-week randomized controlled trial. Its effect on BP and other outcomes shall be confirmed in a larger sample.