Next‐generation sequencing in two cases of de novo acute basophilic leukaemia

Acute basophilic leukaemia (ABL) is a rare subtype of acute myeloid leukaemia (AML); therefore, few data are available about its biology. Herein, we analysed two ABL patients using flow cytometry and next-generation sequencing (NGS). Two cell populations were detected by flow cytometry in both patients. In Case no. 1, blasts (CD34⁺, CD203c⁻, CD117⁺, CD123dim⁺) and basophils (CD34⁻, CD203c⁺, CD117±, CD123⁺) were identified, both of which were found by NGS to harbour the 17p deletion and have loss of heterozygosity of TP53. In Case no. 2, blasts (CD33⁺, CD34⁺, CD123⁻) and basophils (CD33⁺, CD34⁺, CD123⁺) were identified. NGS detected NPM1 mutations in either blasts or basophils, and TET2 in both. These data suggest an overlap of the mutational landscape of ABL and AML, including TP53 and TET2 mutations. Moreover, additional mutations or epigenetic factors may contribute for the differentiation into basophilic blasts

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

RNAmut: robust identification of somatic mutations in acute myeloid leukemia using RNA-sequencing.

Acute myeloid leukemia (AML) is an aggressive malignancy of haematopoietic stem cells driven by a well-defined set of somatic mutations.1,2 Identifying the mutations driving individual cases is important for assigning the patient to a recognized World Health Organisation category, establishing prognostic risk and tailoring post-consolidation therapy.3 As a result, AML research and diagnostic laboratories apply diverse methodologies to detect important mutations and many are introducing next-generation sequencing (NGS) approaches to study extended panels of genes in order to refine genomic classification and prognostic category.1 Besides the implications of these developments on costs, expertise and reliance on commercial providers, they also do not capture gene expression data, which have independent prognostic value that cannot be inferred from somatic mutation profiles. The ability to detect AML gene mutations as well as gene expression profiles from a single assay, could provide a holistic tool that accelerates research, simplifies diagnostic work-up and helps develop integrated algorithms to refine individual patient prognosis. Here, we show that AML RNA sequencing (RNA-seq) data can be used to reliably detect all types of clinically important mutations and develop a bespoke fast and easy-to-use software (RNAmut) for this purpose that can be readily used by teams/laboratories without in-house bioinformatic expertise

Genetic and clinical landscape of breast cancers with germline BRCA1/2 variants

遺伝性乳癌の遺伝学的・臨床学的特徴を解明 --BRCA1/2 変異乳癌は両アレルの不活化の有無により異なった特徴を持つ--. 京都大学プレスリリース. 2020-10-26.The genetic and clinical characteristics of breast tumors with germline variants, including their association with biallelic inactivation through loss-of-heterozygosity (LOH) and second somatic mutations, remain elusive. We analyzed germline variants of 11 breast cancer susceptibility genes for 1, 995 Japanese breast cancer patients, and identified 101 (5.1%) pathogenic variants, including 62 BRCA2 and 15 BRCA1 mutations. Genetic analysis of 64 BRCA1/2-mutated tumors including TCGA dataset tumors, revealed an association of biallelic inactivation with more extensive deletions, copy neutral LOH, gain with LOH and younger onset. Strikingly, TP53 and RB1 mutations were frequently observed in BRCA1- (94%) and BRCA2- (9.7%) mutated tumors with biallelic inactivation. Inactivation of TP53 and RB1 together with BRCA1 and BRCA2, respectively, involved LOH of chromosomes 17 and 13. Notably, BRCA1/2 tumors without biallelic inactivation were indistinguishable from those without germline variants. Our study highlights the heterogeneity and unique clonal selection pattern in breast cancers with germline variants

Clonal evolution and clinical implications of genetic abnormalities in blastic transformation of chronic myeloid leukaemia

Blast crisis (BC) predicts dismal outcomes in patients with chronic myeloid leukaemia (CML). Although additional genetic alterations play a central role in BC, the landscape and prognostic impact of these alterations remain elusive. Here, we comprehensively investigate genetic abnormalities in 136 BC and 148 chronic phase (CP) samples obtained from 216 CML patients using exome and targeted sequencing. One or more genetic abnormalities are found in 126 (92.6%) out of the 136 BC patients, including the RUNX1-ETS2 fusion and NBEAL2 mutations. The number of genetic alterations increase during the transition from CP to BC, which is markedly suppressed by tyrosine kinase inhibitors (TKIs). The lineage of the BC and prior use of TKIs correlate with distinct molecular profiles. Notably, genetic alterations, rather than clinical variables, contribute to a better prediction of BC prognosis. In conclusion, genetic abnormalities can help predict clinical outcomes and can guide clinical decisions in CML

Postazacitidine clone size predicts long-term outcome of patients with myelodysplastic syndromes and related myeloid neoplasms

Azacitidine is a mainstay of therapy for MDS-related diseases. The purpose of our study is to elucidate the effect of gene mutations on hematological response and overall survival (OS), particularly focusing on their post-treatment clone size. We enrolled a total of 449 patients with MDS or related myeloid neoplasms. They were analyzed for gene mutations in pre- (n=449) and post- (n=289) treatment bone marrow samples using targeted-capture sequencing to assess the impact of gene mutations and their post-treatment clone size on treatment outcomes. In Cox proportional hazard modeling, multi-hit TP53 mutation (HR, 2.03; 95% CI, 1.42-2.91; P<.001), EZH2 mutation (HR, 1.71; 95% CI, 1.14-2.54; P=.009), and DDX41 mutations (HR, 0.33; 95% CI, 0.17-0.62; P<.001), together with age, high-risk karyotypes, low platelet, and high blast counts, independently predicted OS. Post-treatment clone size accounting for all drivers significantly correlated with International Working Group (IWG)-response (P<.001, trend test), except for that of DDX41-mutated clones, which did not predict IWG-response. Combined, IWG-response and post-treatment clone size further improved the prediction of the original model and even that of a recently proposed molecular prediction model, IPSS-M (c-index, 0.653 vs 0.688; P<.001, likelihood ratio test). In conclusion, evaluation of post-treatment clone size, together with pre-treatment mutational profile as well as IWG-response have a role in better prognostication of azacitidine-treated myelodysplasia patients