High-Sensitivity Analysis of Cyanide by Capillary Electrophoresis with Fluorescence Detection

金沢大学大学院自然科学研究科物質情報解析金沢大学工学部A capillary electrophoretic method for a high-sensitivity analysis of cyanide has been developed. Cyanide was derivatized with 2,3-naphthalenedialdehyde and taurine to give a fluorescent product of 1-cyanobenz[f]isoindole. This compound was detected with high sensitivity by fluorescence detection. The detection limit was 0.1 ng/mL, and the calibration curve was linear over the range 0.1 - 200 ng/mL. The precision of the migration time of within-run assays (n = 6) of 1 ng/mL cyanide standard solution was 0.14%. The precision of the peak area for the same runs was 1.0%. This method was applicable to blood analysis. Detection of the cyanide derivative by UV was also examined

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-Prkdcscidll2rgtm1Sug/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

Alteration of the immune environment in bone marrow from children with recurrent B cell precursor acute lymphoblastic leukemia

Due to the considerable success of cancer immunotherapy for leukemia, the tumor immune environment has become a focus of intense research; however, there are few reports on the dynamics of the tumor immune environment in leukemia. Here, we analyzed the tumor immune environment in pediatric B cell precursor acute lymphoblastic leukemia by analyzing serial bone marrow samples from nine patients with primary and recurrent disease by mass cytometry using 39 immunophenotype markers, and transcriptome analysis. High-dimensional single-cell mass cytometry analysis elucidated a dynamic shift of T cells from naïve to effector subsets, and clarified that, during relapse, the tumor immune environment comprised a T helper 1-polarized immune profile, together with an increased number of effector regulatory T cells. These results were confirmed in a validation cohort using conventional flow cytometry. Furthermore, RNA transcriptome analysis identified the upregulation of immune-related pathways in B cell precursor acute lymphoblastic leukemia cells during relapse, suggesting interaction with the surrounding environment. In conclusion, a tumor immune environment characterized by a T helper 1-polarized immune profile, with an increased number of effector regulatory T cells, could contribute to the pathophysiology of recurrent B cell precursor acute lymphoblastic leukemia. This information could contribute to the development of effective immunotherapeutic approaches against B cell precursor acute lymphoblastic leukemia relapse

ACTN1 Mutations Cause Congenital Macrothrombocytopenia

Congenital macrothrombocytopenia (CMTP) is a heterogeneous group of rare platelet disorders characterized by a congenital reduction of platelet counts and abnormally large platelets, for which CMTP-causing mutations are only found in approximately half the cases. We herein performed whole-exome sequencing and targeted Sanger sequencing to identify mutations that cause CMTP, in which a dominant mode of transmission had been suspected but for which no known responsible mutations have been documented. In 13 Japanese CMTP-affected pedigrees, we identified six (46%) affected by ACTN1 variants cosegregating with CMTP. In the entire cohort, ACNT1 variants accounted for 5.5% of the dominant forms of CMTP cases and represented the fourth most common cause in Japanese individuals. Individuals with ACTN1 variants presented with moderate macrothrombocytopenia with anisocytosis but were either asymptomatic or had only a modest bleeding tendency. ACTN1 encodes α-actinin-1, a member of the actin-crosslinking protein superfamily that participates in the organization of the cytoskeleton. In vitro transfection experiments in Chinese hamster ovary cells demonstrated that altered α-actinin-1 disrupted the normal actin-based cytoskeletal structure. Moreover, transduction of mouse fetal liver-derived megakaryocytes with disease-associated ACTN1 variants caused a disorganized actin-based cytoskeleton in megakaryocytes, resulting in the production of abnormally large proplatelet tips, which were reduced in number. Our findings provide an insight into the pathogenesis of CMTP

Integrated genetic and epigenetic analysis defines novel molecular subgroups in rhabdomyosarcoma.

横紋筋肉腫におけるゲノム・エピゲノム異常の全体図を解明 -横紋筋肉腫を4群に分類-. 京都大学プレスリリース. 2015-07-03.Rhabdomyosarcoma (RMS) is the most common soft-tissue sarcoma in childhood. Here we studied 60 RMSs using whole-exome/-transcriptome sequencing, copy number (CN) and DNA methylome analyses to unravel the genetic/epigenetic basis of RMS. On the basis of methylation patterns, RMS is clustered into four distinct subtypes, which exhibits remarkable correlation with mutation/CN profiles, histological phenotypes and clinical behaviours. A1 and A2 subtypes, especially A1, largely correspond to alveolar histology with frequent PAX3/7 fusions and alterations in cell cycle regulators. In contrast, mostly showing embryonal histology, both E1 and E2 subtypes are characterized by high frequency of CN alterations and/or allelic imbalances, FGFR4/RAS/AKT pathway mutations and PTEN mutations/methylation and in E2, also by p53 inactivation. Despite the better prognosis of embryonal RMS, patients in the E2 are likely to have a poor prognosis. Our results highlight the close relationships of the methylation status and gene mutations with the biological behaviour in RMS

Differences in Trocar Positioning within the Vertebral Body Using Two Different Positioning Methods: Effect on Trainee Performance

Purpose. To evaluate the educational effect of the Japanese Society of Interventional Radiology 7th Academic Summer Seminar from a technical perspective. Materials and Methods. Nineteen trainees participated in the seminar. The seminar consisted of vertebroplasty trainings using swine with the single-plane landmark method and with the ISOcenter Puncture (ISOP) method. All trainees were advised by an instructor as they operated the instruments and punctured the vertebra. For each trainee, the accuracy in the final position of the needle tip of the initial puncture in each swine training was evaluated. Results. Error in the final position of the needle tip of ≥5 mm from the target puncture site occurred in the lateral direction in 42% (8/19) of trainees with the landmark method and 5% (1/19) with the ISOP method. No error ≥5 mm occurred in the vertical or anteroposterior directions. In terms of puncture accuracy, error in the lateral direction was significantly lower with the ISOP method than with the landmark method (2.2 ± 1.5 mm versus 5.6 ± 3.2 mm). Conclusion. This seminar was effective training for trocar placement for beginners. The puncture was more accurate with the ISOP method than with the landmark method

Combined landscape of single-nucleotide variants and copy number alterations in clonal hematopoiesis

クローン性造血の臨床予後への影響を解明 --遺伝子変異とコピー数異常の統合的な知見--. 京都大学プレスリリース. 2021-07-09.Clonal hematopoiesis (CH) in apparently healthy individuals is implicated in the development of hematological malignancies (HM) and cardiovascular diseases. Previous studies of CH analyzed either single-nucleotide variants and indels (SNVs/indels) or copy number alterations (CNAs), but not both. Here, using a combination of targeted sequencing of 23 CH-related genes and array-based CNA detection of blood-derived DNA, we have delineated the landscape of CH-related SNVs/indels and CNAs in 11, 234 individuals without HM from the BioBank Japan cohort, including 672 individuals with subsequent HM development, and studied the effects of these somatic alterations on mortality from HM and cardiovascular disease, as well as on hematological and cardiovascular phenotypes. The total number of both types of CH-related lesions and their clone size positively correlated with blood count abnormalities and mortality from HM. CH-related SNVs/indels and CNAs exhibited statistically significant co-occurrence in the same individuals. In particular, co-occurrence of SNVs/indels and CNAs affecting DNMT3A, TET2, JAK2 and TP53 resulted in biallelic alterations of these genes and was associated with higher HM mortality. Co-occurrence of SNVs/indels and CNAs also modulated risks for cardiovascular mortality. These findings highlight the importance of detecting both SNVs/indels and CNAs in the evaluation of CH

Fusion partner–specific mutation profiles and KRAS mutations as adverse prognostic factors in MLL-rearranged AML

急性骨髄性白血病の予後を予測する新規マーカーを発見 --リスクに応じた適切な治療につながる可能性--. 京都大学プレスリリース. 2020-10-02.Mixed-lineage leukemia (MLL) gene rearrangements are among the most frequent chromosomal abnormalities in acute myeloid leukemia (AML). MLL fusion patterns are associated with the patient’s prognosis; however, their relationship with driver mutations is unclear. We conducted sequence analyses of 338 genes in pediatric patients with MLL-rearranged (MLL-r) AML (n = 56; JPLSG AML-05 study) alongside data from the TARGET study’s pediatric cohorts with MLL-r AML (n = 104), non–MLL-r AML (n = 581), and adult MLL-r AML (n = 81). KRAS mutations were most frequent in pediatric patients with high-risk MLL fusions (MLL-MLLLT10, MLL-MLLT4, and MLL-MLLT1). Pediatric patients with MLL-r AML (n = 160) and a KRAS mutation (KRAS-MT) had a significantly worse prognosis than those without a KRAS mutation (KRAS-WT) (5-year event-free survival [EFS]: 51.8% vs 18.3%, P < .0001; 5-year overall survival [OS]: 67.3% vs 44.3%, P = .003). The adverse prognostic impact of KRAS mutations was confirmed in adult MLL-r AML. KRAS mutations were associated with adverse prognoses in pediatric patients with both high-risk (MLLT10+MLLT4+MLLT1; n = 60) and intermediate-to-low–risk (MLLT3+ELL+others; n = 100) MLL fusions. The prognosis did not differ significantly between patients with non–MLL-r AML with KRAS-WT or KRAS-MT. Multivariate analysis showed the presence of a KRAS mutation to be an independent prognostic factor for EFS (hazard ratio [HR], 2.21; 95% confidence interval [CI], 1.35-3.59; P = .002) and OS (HR, 1.85; 95% CI, 1.01-3.31; P = .045) in MLL-r AML. The mutation is a distinct adverse prognostic factor in MLL-r AML, regardless of risk subgroup, and is potentially useful for accurate treatment stratification. This trial was registered at the UMIN (University Hospital Medical Information Network) Clinical Trials Registry (UMIN-CTR; http://www.umin.ac.jp/ctr/index.htm) as #UMIN000000511

Identification of the ultrahigh-risk subgroup in neuroblastoma cases through DNA methylation analysis and its treatment exploiting cancer metabolism

神経芽腫の新たな診断法と治療戦略を創出 --がん細胞の生存戦略「がん代謝」を逆用する--. 京都大学プレスリリース. 2022-11-02.Neuroblastomas require novel therapies that are based on the exploitation of their biological mechanism. To address this need, we analyzed the DNA methylation and expression datasets of neuroblastomas, extracted a candidate gene characterizing the aggressive features, and conducted functional studies. Based on the DNA methylation data, we identified a subgroup of neuroblastoma cases with 11q loss of heterozygosity with extremely poor prognosis. PHGDH, a serine metabolism-related gene, was extracted as a candidate with strong expression and characteristic methylation in this subgroup as well as in cases with MYCN amplification. PHGDH inhibition suppressed neuroblastoma cell proliferation in vitro and in vivo, indicating that the inhibition of serine metabolism by PHGDH inhibitors is a therapeutic alternative for neuroblastoma. Inhibiting the arginine metabolism, which is closely related to serine metabolism using arginine deiminase, had a combination effect both in vitro and in vivo, especially on extracellular arginine-dependent neuroblastoma cells with ASS1 deficiency. Expression and metabolome analyses of post-dose cells confirmed the synergistic effects of treatments targeting serine and arginine indicated that xCT inhibitors that inhibit cystine uptake could be candidates for further combinatorial treatment. Our results highlight the rational therapeutic strategy of targeting serine/arginine metabolism for intractable neuroblastoma