The outcome of treatment-refractory and/or relapsed pediatric T cell acute lymphoblastic leukemia (T-ALL) is extremely poor, and the genetic basis for this is not well understood. Here we report comprehensive profiling of 121 cases of pediatric T-ALL using transcriptome and/or targeted capture sequencing, through which we identified new recurrent gene fusions involving SPI1 (STMN1-SPI1 and TCF7-SPI1). Cases positive for fusions involving SPI1 (encoding PU.1), accounting for 3.9% (7/181) of the examined pediatric T-ALL cases, showed a double-negative (DN; CD4CD8) or CD8 single-positive (SP) phenotype and had uniformly poor overall survival. These cases represent a subset of pediatric T-ALL distinguishable from the known T-ALL subsets in terms of expression of genes involved in T cell precommitment, establishment of T cell identity, and post-β-selection maturation and with respect to mutational profile. PU.1 fusion proteins retained transcriptional activity and, when constitutively expressed in mouse stem/progenitor cells, induced cell proliferation and resulted in a maturation block. Our findings highlight a unique role of SPI1 fusions in high-risk pediatric T-ALL.

The prognosis of infants with acute lymphoblastic leukemia (ALL), particularly those with KMT2A gene rearrangement (KMT2A-r), is dismal. Continuous efforts have been made in Japan to investigate the role of stem cell transplantation (HSCT) for infants with KMT2A-r ALL, but improvement in outcomes was modest. In the Japanese Pediatric Leukemia/Lymphoma Study Group trial MLL-10 (registered at umin.ac.jp as UMIN000004801), infants with ALL were stratified into three risk groups (low-risk, LR; intermediate-risk, IR; high-risk, HR) according to KMT2A status, age, and presence of central nervous system leukemia. A modified Children's Oncology Group AALL0631 chemotherapy with addition of high-dose cytarabine in early intensification was introduced to KMT2A-r patients, and the option of HSCT was restricted to HR patients. The role of minimal residual disease (MRD) was also evaluated. Ninety eligible infants were stratified into LR (N=15), IR (N=19), or HR (N=56). The 3-year event-free survival (EFS) rate (standard error) for patients with KMT2A-r ALL (IR+HR) was 66.2% (5.6%) and 93.3% (6.4%) for those with KMT2A-g ALL (LR). The 3-year EFS rate was 94.4% (5.4%) for IR and 56.6% (6.8%) for HR patients. In multivariable analysis, female sex and MRD ≧0.01% at end of early consolidation were significant poor prognostic factors. Risk stratification and introduction of intensive chemotherapy in the current study were effective and able to eliminate HSCT for a subset of infants with KMT2A-r ALL. Early clearance of MRD seems to have translated into favorable outcomes and should be incorporated into risk stratifications in future trials.

CT designed the research, analyzed the data and wrote the paper; IS supervised the work; PVR, SC, RP and CDB performed experiments; GZ provided AML cell lines, reagents and analyzed the data; SC and DB commented on the paper. SC and RF provided AML samples. AA provided TALL samples. CT, PVR, SC assembled the figures. AUTHOR CONTRIBUTIONS CT designed the research, analyzed the data and wrote the paper; IS supervised the work; PVR, SC, RP and CDB performed experiments; GZ provided AML cell lines, reagents and analyzed the data; SC and DB commented on the paper. SC and RF provided AML samples. AA provided TALL samples. CT, PVR, SC assembled the figures.

Although hepatoblastoma is the most common pediatric liver cancer, its genetic heterogeneity and therapeutic targets are not well elucidated. Therefore, we conducted a multiomics analysis, including mutatome, DNA methylome, and transcriptome analyses, of 59 hepatoblastoma samples. Based on DNA methylation patterns, hepatoblastoma was classified into three clusters exhibiting remarkable correlation with clinical, histological, and genetic features. Cluster F was largely composed of cases with fetal histology and good outcomes, whereas clusters E1 and E2 corresponded primarily to embryonal/combined histology and poor outcomes. E1 and E2, albeit distinguishable by different patient age distributions, were genetically characterized by hypermethylation of the HNF4A/CEBPA-binding regions, fetal liver-like expression patterns, upregulation of the cell cycle pathway, and overexpression of NQO1 and ODC1. Inhibition of NQO1 and ODC1 in hepatoblastoma cells induced chemosensitization and growth suppression, respectively. Our results provide a comprehensive description of the molecular basis of hepatoblastoma and rational therapeutic strategies for high-risk cases.

Programmed death 1 (PD‐1)/programmed death ligand 1 (PD‐L1) pathway blockade has become a promising therapeutic target in adult cancers. We evaluated PD‐L1 expression and tumor‐infiltrating CD8+ T cells in formalin‐fixed, paraffin‐embedded tumor specimens from 53 untreated pediatric patients with eight cancer types: neuroblastoma, extracranial malignant germ cell tumor, hepatoblastoma, germinoma, medulloblastoma, renal tumor, rhabdomyosarcoma, and atypical teratoid/rhabdoid tumor. One rhabdomyosarcoma with the shortest survival exhibited membranous PD‐L1 expression and germinoma contained abundant tumor‐infiltrating CD8+ T cells and PD‐L1‐positive macrophages. The PD‐1/PD‐L1 pathway tended to be inactive in pediatric cancers.