ALBERT

Description: To elucidate the molecular pathogenesis of pediatric germ cell tumors (GCTs), we performed DNA methylation array analysis, whole transcriptome sequencing, targeted capture sequencing, and single-nucleotide polymorphism array analysis using 51 GCT samples (25 female, 26 male), including 6 germinomas, 2 embryonal carcinomas, 4 immature teratomas, 3 mature teratomas, 30 yolk sac tumors, and 6 mixed germ cell tumors. Among the 51 samples, 11 were from infants, 23 were from young children, and 17 were from those aged ≥10 years. Sixteen of the 51 samples developed in the extragonadal regions. Germinomas showed upregulation of pluripotent genes and global hypomethylation. Pluripotent genes were also highly expressed in embryonal carcinomas. These genes may play essential roles in embryonal carcinomas given that their binding sites are hypomethylated. Yolk sac tumors exhibited overexpression of endodermal genes, such as GATA6 and FOXA2, the binding sites of which were hypomethylated. Interestingly, infant yolk sac tumors had different DNA methylation patterns from those observed in older children. Teratomas had higher expression of ectodermal genes, suggesting a tridermal nature. Based on our results, we suggest that KIT, TNFRSF8, and ERBB4 may be suitable targets for the treatment of germinoma, embryonal carcinomas, and yolk sac tumors, respectively.

Description: Introduction TP53 mutations in relapsed cases with pediatric acute lymphoblastic leukemia have been implicated in poor clinical outcomes. However, the prevalence and clinical significance of TP53 mutations at diagnosis have not been fully investigated. Such knowledge is essential for the care of patients, because treatment intensity is tailored to predictive prognosis, where increased attention has been directed toward de-escalation of treatment for the problem of long term effects and second malignancies in childhood cancer survivors. Methods Mutation status of TP53 was detected by targeted-capture sequencing of TP53 coding regions in 1,003 children with B-precursor ALL who had been treated in either of the two prospective clinical trials, JACLS (Japan Association of Childhood Leukemia Study) ALL-02 and TCCSG (Tokyo Children's Cancer Study Group) L04-16. Detection of common fusion genes, including BCR-ABL, ETV6-RUNX1, MLL-AF4, MLL-ENL, MLL-AF9, and TCF3-PBX1, were performed using qPCR assays. We designed SNP baits to analyze copy number status of chromosome 17, and also captured 662 probes tiling the entire IgH enhancer locus to identify IGH-DUX4 rearrangement. Result In total, 36 different non-silent coding TP53 mutations were identified in 30 (3%) patients, including 22 missense, 7 frameshift indel, 5 in-frame indel, and 2 nonsense mutations. All missense mutations were found in the core DNA-binding domain (n=21), except for one mutation, which affected the tetramerization motif. Variant allele frequencies (VAF) of TP53 mutations varied from 3% to 97% with 14 mutations showing 〈 10% VAFs. Showing a significant correlation with mutated TP53 (Odds ratio 20: 95%CI 6.4-61, P

Description: Background Acute lymphoblastic leukemia (ALL) in Down syndrome (DS) have uncommon genetic alterations such as mutations of JAK2, RAS, and overexpressions of CRLF2. These findings suggest DS-ALL may have unique biological features compared with non-DS-ALL. While recent studies implicated HMGN1 or DYRK1A in chromosome 21 were associated with molecular pathogenesis of DS-ALL, it remains to be elucidated what predispose DS children to develop ALL. Materials and Methods We performed whole transcriptome sequencing, targeted deep sequencing, and SNP array analysis in 25 DS-ALL samples, which included four ETV6-RUNX1 fusions and one high hyperdiploid. To compare with DS-ALL, we also performed whole transcriptome sequencing and whole exome sequencing to 118 non-DS-ALL samples, which included several subtypes such as ETV6-RUNX1 or BCR-ABL1. To cluster gene expression profiling, we applied the hierarchical clustering method. The detection of Ph-like signatures was performed by the hierarchical clustering by the gene set reported by Harvey. Results In expression analysis, we identified 19 fusions in 25 DS-ALL samples. These fusions included 15 recurrent fusions in pediatric BCP-ALL and 4 novel fusions, which including SSBP3-DHCR24, PDGFA-TTYH3, and NIN-NDUFA6. In novel fusions, PDGFA-TTYH3 fusions were detected in two DS-ALL samples. The hierarchical clustering analysis (Figure 1) combining 25 DS-ALL with 123 non-DS ALL samples. In our cohort, we defined samples with PAX5 alteration only such as a mutation or fusion as PAX5-altered. This clustering revealed ALL samples were divided into six clusters (cluster E1 to E6). Among six clusters, DS-ALL samples were divided into four clusters. In these four clusters, chi-square test revealed the significant enrichment of DS-ALL in E6 cluster. Importantly, our expression analysis revealed DS-ALL samples were highly heterogeneous and had the same expression pattern corresponding to each subtype same as non-DS-ALL. Cluster E3 included most samples with PAX5 fusions. All samples with ETV6-RUNX1 fusions were classified into cluster E4. Most samples of high hyperdiploid were classified into cluster E5. Cluster E6 was characterized by BCR-ABL1 fusions and Ph-like signatures. We detected 21 samples had Ph-like signatures, which included seven DS-ALL samples and 14 non-DS-ALL samples. Though we also analyzed differentially expressed genes between DS-ALL and non-DS-ALL, no genes on chromosome 21 such as HNGN1 or DYRK1A was significantly expressed. To investigate a relation between expression and genomic status, we further searched mutational analysis and copy number analysis (Figure 2). In 25 DS-ALL samples, six samples revealed JAK2 mutations and CRLF2 fusions. Interestingly, all of these six samples had Ph-like signatures. In cluster E5, one non-DS-ALL sample revealed JAK2 mutation and CRLF2 fusion and this particular sample was expected to have the Ph-like signature. To detect other Ph-like samples, we performed hierarchical clustering of 143 ALL samples based on the genes with a significantly (adjusted P value

Description: [Background] Mixed lineage leukemia (MLL also called KMT2A) rearrangement-positive leukemia is one of the most aggressive types of leukemia. It is diagnosed predominantly in infants and typically shows a multi-lineage phenotype. Since current chemotherapy fails in more than 50% of infantile acute lymphoblastic leukemia (ALL) with MLL-rearrangement, a better understanding of biological features of the disease is important in order to develop more specific and successful treatment strategies. Despite the evident biological and clinical significance of MLL-rearrangement, it has also been shown in vitro and in vivo that MLL-rearrangement is not sufficient to induce full leukemic transformation, indicating additional hits are required for complete leukemogenesis. Recently, sequencing-based genome-wide studies have suggested collaborative involvement of RAS-PI3K pathway in infant leukemogenesis through identification of recurrent mutations affecting RAS-PI3K pathway genes, in infant ALL with or without MLL-rearrangement. However, genetic basis of infant ALL with MLL-rearrangement are not fully elucidated. [Materials and Methods] Here, we performed whole exome sequencing in leukemia samples with matched peripheral blood T lymphocyte from 46 cases of infant ALL including 3 cases with germline MLL. Our cohort included samples from the Infant Leukemia Sub-committee, Japan Children's Cancer Group. For mapping and mutation calling, we used publicly available "Genomon Pipeline". [Results] In total, 198 somatic non-silent mutations (4.3 mutations per case) were identified, including 125 missense, 4 nonsense, 8 splice site mutations, 49 frameshift indels, and 12 in-frame indels. Thirteen genes were recurrently affected, among which RAS-PI3K pathway mutations were the most frequent: KRAS (10/46 cases, 21.7%), NRAS (4/46 cases, 8.7%) and PTPN11 (3/46 cases, 6.5%). In addition, novel recurrent mutations were detected in HSP90AB1 (5/46 cases, 10.9%), MAPRE3 (2/46 cases, 4.3%) and SRCAP (2/46 cases, 4.3%), as well as known candidate driver mutations in FLT3 (7/46 cases, 15.2%), TP53 (3/46 cases, 6.5%), CHD4 (2/46 cases, 4.3%) and PAX5 (2/46 cases, 4.3%). HSP90AB1 is a member of Heat shock protein90 (Hsp90) family that encodes a group of molecular chaperones involved in stabilization and activation of multiple oncogenic proteins and pathways. Overexpression of Hsp90 is observed in ALL, chronic myeloid/lymphocytic leukemia and several non-hematological malignancies. Another recurrently mutated gene, SRCAP, encodes the core component of chromatin-remodeling Snf2-related CREBBP activator protein complex. Recurrent mutations in this gene have recently been reported in FLT3-ITD positive acute myeloid leukemia with significantly high co-occurrence of MLL3 mutations. Overall, RAS-PI3K pathway mutations were detected in 19/46 cases of infant ALL (41.3%). In our cohort, none of 3 germline MLL cases contained RAS-PI3K pathway mutations; one case had a PAX5 mutation, another had a SRCAP mutation, and the other had no candidate driver mutations. In the germline MLL case without candidate drivers, G-banding had identified a structural abnormality, t(5;15)(p15;q11.2), that is rare but reported to be predominant in infant ALL rather than other hematological/non-hematological malignancies. [Conclusion] Our results demonstrated that aberrations of cell proliferation signaling, transcription factors, as well as cell cycle/epigenetic regulators are co-operative oncogenic events in MLL-rearrangement cases. The mutation spectrum is similar to myeloid malignancies rather than lymphoid cancers. Intriguingly, we identified novel recurrent mutations in the oncogenic proteins, such as HSP90AB1, MAPRE3, and SRCAP,suggesting that these are potentially targetable by small-molecule therapy. In addition, we also illustrated the genetic differences between MLL-rearrangement and germline MLL. Thus, our mutational landscape provides a novel insight into the molecular mechanisms of infant ALL and may contribute to improving the clinical outcome of infant patients suffering from the intractable ALL. Disclosures Ogawa: Kan research institute: Consultancy, Research Funding; Takeda Pharmaceuticals: Consultancy, Research Funding; Sumitomo Dainippon Pharma: Research Funding.

Description: T-cell acute lymphoblastic leukemia/lymphoma (T-ALL/LBL) accounts for 10% to 15% of newly diagnosed cases of childhood acute lymphoblastic leukemia (ALL), arising from the malignant transformation of hematopoietic progenitors primed toward T cell development, as result of a multistep oncogenic process. However, since the prognostic significance of these genetic alterations in pediatric T-ALL is not clear, genetic basis which contributes aggressive phenotype or progression of pediatric T-ALL is still to be elucidated. Therefore, to discover driver genetic events, which involved in the aggressive phenotype of pediatric T-ALL and to identify it's novel prognostic markers, we performed integrated genetic analysis in a large cohort of T-ALL case. Our cohorts included samples from Tokyo Children's Cancer Study Group (TCCSG) and Japan Association of Childhood Leukemia Study (JACLS). Whole transcriptome sequencing (WTS) was performed in 123 cases. Representative recurrent fusion genes were as follows, SIL-TAL1 (n=25), MLL-ENL (n=5), PICALM-MLLT10 (n=5), and NUP214-ABL1 (n=2). Intriguingly, novel recurrent in-frame SPI1 fusions (STMN1-SPI1 n=2; TCF7-SPI1 n=5) were detected, and RT-PCR analysis in additional 60 cases revealed other 2 TCF7-SPI1 fusions. Thus, SPI1 fusions accounted for 4% of pediatric T-ALL/LBL. Expression data of WTS revealed cases with SPI1 fusion showed significantly higher expression of SPI1 compared to cases without SPI1 fusion, implicating that aberrant high expression of SPI1 involved in leukemogenesis. To address the functional activities of SPI1 fusions, we performed luciferase assay using the reporter vector contained the CSF1 promoter region with SPI1 binding site. Transient transfection of Hela cells with the SPI1 fusions expression vectors as well as the wild type SPI1 expression vector showed strikingly high levels of transcription of the reporter genes, as compared to transfection with the empty expression vector, indicating that both SPI1 fusions have transcriptional activities. Next, to analyze the leukemogenic potential of SPI1 fusions in vitro, we transduced fusions cDNA into mouse double negative T-cells. Since p16(CDKN2A) is frequently silenced in T-ALL, we also used p16 null double negative T-cells. Both wild-type and p16 null double negative T-cells expressing SPI1 fusions showed increased cell proliferation compared to the MOCK cells. We further evaluated the impact of SPI1 fusions on T cells differentiation. TCF7-SPI1 or MOCK vector was transduced mononuclear cells isolated from mouse bone marrow. These cells were cultured under stimulating factors, such as IL6 and TPO for 3 days, and then transplanted into the irradiated mouse. Subsequently, 6 week after transplantation, FACS analysis was performed. Of interest, significantly high population of cells expressing TCF7-SPI1 was observed in the immature single positive stage, implicating that their differentiation was impaired at the pre-T cell stage. These results indicate that novel SPI1 fusions have a potential leukemogenic effect in pediatric T-ALL. We defined SPI1 overexpression cases as outliers of SPI1 expression, resulting in extremely poor prognosis (log-rank p = 1.9 ×10-6). Of note, significant poor outcome was confirmed by univariate and multivariate analysis in cases with SPI1 overexpression cases (log-rank p = 9.3 ×10-6, and p = 2.0 ×10-6, respectively). In conclusion, SPI1 fusions expressing cells expanded and they remained at an immature stage, implicating a potential leukemogenic activity of these fusions. Not only the cases with SPI1 fusions, but also the cases with high SPI1 expression without fusions showed extremely poor prognosis, suggesting the prognostic value of aberrant SPI1 expression in pediatric T-ALL. Although it remains unclear, why cases with SPI1 fusions/high SPI1 expression have a poor prognosis, our results indicate that these cases are genetically distinct subgroup from other pediatric T-ALL. Disclosures Kataoka: Kyowa Hakko Kirin: Honoraria; Yakult: Honoraria; Boehringer Ingelheim: Honoraria. Ogawa:Kan research institute: Consultancy, Research Funding; Takeda Pharmaceuticals: Consultancy, Research Funding; Sumitomo Dainippon Pharma: Research Funding.