Masanori Yoshida

BCOR and BCORL1 (BCOR/L1 ) mutations are observed in both myeloid neoplasms (MN) and bone marrow failure syndromes (BMF), but their clinical significance may differ. We analyzed 11 340 patients and identified 418 BCOR/L1 ‐mutated patients, focusing on differences between MN and BMF. Compared with MN, BMF was associated with younger age, a higher frequency of normal karyotype, a greater proportion of missense mutations, and significantly lower variant allele frequencies (VAFs). We next compared BCOR/L1 ‐mutated cases with wild‐type cases. In MN, BCOR/L1 mutations were associated with older age, inferior survival, and a higher number of co‐occurring mutations, whereas in BMF, they were associated with a higher prevalence of PNH clones. Single‐cell DNA sequencing and VAF‐based analysis demonstrated that BCOR/L1 mutations were usually secondary in MN but primary events in BMF. Longitudinal sequencing revealed that BCOR/L1 ‐mutant clones often contracted or disappeared over time, even in progression, suggesting limited clonal fitness of BCOR/L1 mutations themselves; instead, progression was associated with increased mutational burden, particularly in RUNX1 . Prognostically, BCOR/L1 mutations were associated with inferior survival in MN, whereas in BMF they were not associated with prognosis and tended to correlate with a favorable response to immunosuppressive therapy. RNA‐sequencing analyses showed reduced BCOR/L1 expression in BMF, and BCOR knockout was associated with reduced HLA class I expression. Our findings suggest that BCOR/L1 mutations are secondary events in MN and co‐occurring mutations may influence disease progression and prognosis, whereas in BMF they appear to be ancestral events acquired independently of progression to MN, potentially through immune privilege‐related mechanisms.

Aplastic anemia (AA) results from T-cell-mediated destruction of hematopoietic stem and progenitor cells (HSPCs), driving clonal hematopoiesis via loss of human leukocyte antigen (HLA) risk alleles (HLA loss-of-function mutations or uniparental disomy 6p, UPD6p), paroxysmal nocturnal hemoglobinuria and clonal hematopoiesis of indeterminate potential (CHIP) mutations. Here genomic profiling of 619 patients with AA revealed clonal hematopoiesis in 69% of cases, with ASXL1, BCOR and BCORL1 identified as the most frequent CHIP mutations in pediatric cases. Single-cell multi-omics analysis of 304,902 cells from 48 samples uncovered complex branching clonal architecture, with a median of three HLA loss events per patient, converging to inactivate HLA risk alleles. Single-cell whole-genome sequencing (WGS) resolved up to 15 HLA loss clones per patient and phylogenetic reconstruction indicated that these clones originated years before diagnosis. Long-read WGS precisely mapped UPD6p breakpoints and HLA methylation. HLA loss conferred a protective effect against CHIP, evidenced by their near-absent co-occurrence. Longitudinal single-cell analysis demonstrated that long-lived clones were enriched in the CD34⁺ HSPC compartment. These findings reveal parallel evolutionary pathways used by hematopoietic cells to evade immune attack.

6-Mercaptopurine (6-MP) is widely used for the treatment of paediatric leukaemia and lymphoma. Recently, germline variants in the NUDT15 gene have been identified as one of the major genetic causes for 6-MP-associated adverse effects such as myelosuppression. Patients with hypomorphic NUDT15 variants accumulate excessive levels of DNA-incorporated thioguanine in white blood cells, resulting in severe myelosuppression. Although preclinical studies suggest that these variants may influence the protein stability of NUDT15, this has not been directly characterised in patients. In this study, we report the development of a series of novel monoclonal antibodies against NUDT15, using which we quantitatively assessed NUDT15 protein levels in 37 patients with acute lymphoblastic leukaemia treated with 6-MP, using sandwich enzyme-linked immunosorbent assay (ELISA). The NUDT15 genotype was highly correlated with its protein levels (p < 0.0001), with homozygous and compound heterozygous patients showing exceedingly low NUDT15 expression. There was a positive correlation between NUDT15 protein level and 6-MP tolerance (r = 0.631, p < 0.0001). In conclusion, our results point to low NUDT15 protein abundance as the biochemical basis for NUDT15-mediated 6-MP intolerance, thus providing a phenotypic readout of inherited NUDT15 deficiency.

The effect of genetic variation on second malignant neoplasms (SMNs) remains unclear. First, we identified the pathogenic germline variants in cancer-predisposing genes among 15 children with SMNs after childhood leukemia/lymphoma using whole-exome sequencing. Because the prevalence was low, we focused on the association between SMNs and NUDT15 in primary acute lymphoblastic leukemia (ALL) cases. NUDT15 is one of the 6-mercaptopurine (6-MP) metabolic genes, and its variants are common in East Asian individuals. The prevalence of NUDT15 hypomorphic variants was higher in patients with SMNs (n = 14; 42.9%) than in the general population in the gnomAD database (19.7%; P = .042). In the validation study with a cohort of 438 unselected patients with ALL, the cumulative incidence of SMNs was significantly higher among those with (3.0%; 95% confidence interval [CI], 0.6% to 9.4%) than among those without NUDT15 variants (0.3%; 95% CI, 0.0% to 1.5%; P = .045). The 6-MP dose administered to patients with ALL with a NUDT15 variant was higher than that given to those without SMNs (P = .045). The 6-MP–related mutational signature was observed in SMN specimens after 6-MP exposure. In cells exposed to 6-MP, a higher level of 6-MP induced DNA damage in NUDT15-knockdown induced pluripotent stem cells. Our study indicates that NUDT15 variants may confer a risk of SMNs after treatment with 6-MP in patients with ALL.

抗血小板薬は脳梗塞，心筋梗塞，末梢動脈血栓症など動脈血栓症の治療や予防に使用されている。外科手術時に抗血小板薬を血栓・塞栓症の発症を考慮し継続するのか，大出血のリスクのため中止するかについては，明確な基準はない。今回，抗血小板薬内服継続下での頸部郭清術について臨床的検討を行ったので報告する。 対象は2010年7月から2020年12月までに口腔癌原発手術後に頸部リンパ節後発転移を認め，当科にて片側全頸部郭清術を単独で施行した症例の中で，条件を満たした23例とした。対象を抗血小板薬内服群と非内服群に分けて後方視的に比較検討した。内服群は6例，非内服群は17例であった。術中出血量，術後ドレーン量，術後Hb，RBC変化率は両群間で有意差はなかった。両群において術後出血や術後出血性合併症，血栓性合併症の発症はなかった。 本研究において抗血小板薬内服継続下でも頸部郭清術を施行できることが示された。しかしながら，頸部郭清術における術後出血は気道閉塞により生命の危険を招く可能性があり，抗血小板薬を継続する場合は，術中の慎重な止血操作や術後の厳重な管理が必須であると考えられた。また，本研究は症例数が少ないため今後は多施設共同で症例を蓄積し，検討を行う必要があると考えられた。

Summary: Monosomy 7 (−7) occurs in various types of paediatric myeloid disorders and has a poor prognosis. Recent studies have demonstrated that patients with germline gain‐of‐function SAMD9/9L variants and loss‐of‐function GATA2 variants are prone to developing myelodysplastic syndrome (MDS) associated with −7. However, the prevalence of the genetic variants among paediatric haematologic disorders with −7 is unknown. The present study screened germline variants of GATA2 and SAMD9/9L in 25 patients with various types of paediatric haematological disorders associated with −7. The diagnoses of the 25 patients included MDS (n = 10), acute myeloid leukaemia (AML) and myeloid sarcomas (n = 9), juvenile myelomonocytic leukaemia (n = 3) and other disorders (n = 3). Seven patients with a germline pathogenic GATA2 variant were found. For SAMD9/9L screening, next‐generation sequencing was used to detect low‐abundance variants and found four novel germline variants. Functional analysis revealed that three out of the four variants showed growth‐restricting capacity in vitro and thus, were judged to be pathogenic. Cases with GATA2 mutation tended to be older, compared to those with SAMD9/9L mutations. In conclusion, GATA2 and SAMD9/9L were sequenced in 25 patients with paediatric haematologic disorders associated with −7, and 40% of them were found to have some pathogenic germline variants in the three genes.

Intensive analysis of the SMARCB1 gene in malignant rhabdoid tumors (MRT) revealed eight of 16 patients with constitutional genetic variants. Three patients had mosaicism of deletion/variant of the SMARCB1 gene, which conventional methods might overlook. The prevalence of cancer predisposition in MRT may thus be higher than previously reported.

Acute promyelocytic leukemia (APL) is cytogenetically characterized by the t(15;17) (q24;q21), although cases without this translocation exist. These cases are referred to as “cryptic” or “masked” translocations. Additionally, fewer than 5% of APL cases have another partner gene fused to the RARA gene. The TBL1XR1‐RARA fusion gene has recently been reported as a novel RARA‐associated fusion gene. We report a case with TBL1XR1‐RARA and a masked translocation that was not detected by conventional tests for RARA‐associated translocations. Three‐year‐old girl was diagnosed with APL based morphological findings, although conventional tests for RARA‐associated chimeric genes were negative. She received all‐trans retinoic acid treatment, but that was not effective. She achieved a complete remission (CR) by conventional multidrug chemotherapy, but had extramedullary relapse 2 years after onset. She underwent cord blood transplantation (CBT) in her second CR and is currently alive. To investigate the underlying pathogenesis of this unique case, we performed whole‐genome sequencing and found a cryptic insertion of RARA gene into the TBL1XR1 gene. The transcript of the chimeric gene, TBL1XR1‐RARA, was confirmed as an in‐frame fusion by RT‐PCR. In conclusion, we found using next‐generation sequencing (NGS) a TBL1XR1‐RARA fusion in a child with variant APL without the classic karyotype. Cryptic insertion could also occur in cases other than APL with PML‐RARA. Variant APL has many variants and NGS analysis should therefore be considered for APL variant cases, even for those without RARA translocation detected by conventional analysis.

Infant acute lymphoblastic leukemia with lysine (K)‐specific methyltransferase 2A (KMT2A) rearrangements usually has a poor prognosis regardless of the fusion partners of KMT2A. However, the prognosis of pediatric acute myeloid leukemia (AML) with KMT2A rearrangements depends on its translocation partners. We herein report the case of a 9‐month‐old boy with a KMT2A‐USP2 fusion, which required diagnosis by whole transcriptome sequencing after the failure of detection of known translocation partners by conventional screening approaches. As this first report of a patient with AML with a KMT2A‐USP2 fusion illustrates, identification of the partners in all patients with KMT2A‐rearranged AML is critical to elucidate the outcomes associated with specific rearrangements and to develop appropriate treatment strategies. Moreover, development of additional methods to detect specific translocation partners of KMT2A and leukemia‐specific targeting drugs is important to improve further the outcomes of KMT2A‐rearranged AML.