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1.
Blood ; 135(1): 41-55, 2020 Jan 02.
Artigo em Inglês | MEDLINE | ID: mdl-31697823

RESUMO

To study the mechanisms of relapse in acute lymphoblastic leukemia (ALL), we performed whole-genome sequencing of 103 diagnosis-relapse-germline trios and ultra-deep sequencing of 208 serial samples in 16 patients. Relapse-specific somatic alterations were enriched in 12 genes (NR3C1, NR3C2, TP53, NT5C2, FPGS, CREBBP, MSH2, MSH6, PMS2, WHSC1, PRPS1, and PRPS2) involved in drug response. Their prevalence was 17% in very early relapse (<9 months from diagnosis), 65% in early relapse (9-36 months), and 32% in late relapse (>36 months) groups. Convergent evolution, in which multiple subclones harbor mutations in the same drug resistance gene, was observed in 6 relapses and confirmed by single-cell sequencing in 1 case. Mathematical modeling and mutational signature analysis indicated that early relapse resistance acquisition was frequently a 2-step process in which a persistent clone survived initial therapy and later acquired bona fide resistance mutations during therapy. In contrast, very early relapses arose from preexisting resistant clone(s). Two novel relapse-specific mutational signatures, one of which was caused by thiopurine treatment based on in vitro drug exposure experiments, were identified in early and late relapses but were absent from 2540 pan-cancer diagnosis samples and 129 non-ALL relapses. The novel signatures were detected in 27% of relapsed ALLs and were responsible for 46% of acquired resistance mutations in NT5C2, PRPS1, NR3C1, and TP53. These results suggest that chemotherapy-induced drug resistance mutations facilitate a subset of pediatric ALL relapses.

2.
Eur J Pediatr ; 2019 Dec 05.
Artigo em Inglês | MEDLINE | ID: mdl-31807902

RESUMO

Juvenile myelomonocytic leukemia (JMML) is a heterogeneous childhood leukemia. The management of patients with JMML requires accurate assessment of genetic and clinical features to help in patient risk stratification. This study aimed to investigate the association between genomic alterations and prognosis in children with JMML. Genomic DNA was extracted from a total of 93 patients with JMML for targeted sequencing. Univariable and multivariable analysis were used to evaluate the correlation between gene mutations and prognosis of the patients. Patients with PTPN11 mutation exhibited significantly lower event-free survival (EFS) compared with non-PTPN11 mutations (P = 0.005). Patients without or with one somatic alteration at diagnosis showed significantly better prognosis in comparison with those with more than two alterations (P = 0.009). PTPN11 mutation with additional alterations showed significantly the poorest outcome in comparison with those with only one non-PTPN11 mutation, only one PTPN11 mutation, and combined mutations without PTPN11, respectively (P < 0.0001).Conclusion: Both PTPN11 mutation and the number of somatic alterations detected at diagnosis are likely to be the major determinant of outcome in JMML. The subgroup of patients with PTPN11 mutation showed the shortest survival which was even worsened when a secondary mutation was present.

3.
Leukemia ; 33(10): 2365-2378, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-30940905

RESUMO

Bone marrow (BM) niche responds to chemotherapy-induced cytokines secreted from acute lymphoblastic leukemia (ALL) cells and protects the residual cells from chemotherapeutics in vivo. However, the underlying molecular mechanisms for the induction of cytokines by chemotherapy remain unknown. Here, we found that chemotherapeutic drugs (e.g., Ara-C, DNR, 6-MP) induced the expression of niche-protecting cytokines (GDF15, CCL3 and CCL4) in both ALL cell lines and primary cells in vitro. The ATM and NF-κB pathways were activated after chemotherapy treatment, and the pharmacological or genetic inhibition of these pathways significantly reversed the cytokine upregulation. Besides, chemotherapy-induced NF-κB activation was dependent on ATM-TRAF6 signaling, and NF-κB transcription factor p65 directly regulated the cytokines expression. Furthermore, we found that both pharmacological and genetic perturbation of ATM and p65 significantly decreased the residual ALL cells after Ara-C treatment in ALL xenograft mouse models. Together, these results demonstrated that ATM-dependent NF-κB activation mediated the cytokines induction by chemotherapy and ALL resistance to chemotherapeutics. Inhibition of ATM-dependent NF-κB pathway can sensitize ALL to chemotherapeutics, providing a new strategy to eradicate residual chemo-resistant ALL cells.

4.
Genome Biol ; 20(1): 50, 2019 03 14.
Artigo em Inglês | MEDLINE | ID: mdl-30867008

RESUMO

BACKGROUND: Sequencing errors are key confounding factors for detecting low-frequency genetic variants that are important for cancer molecular diagnosis, treatment, and surveillance using deep next-generation sequencing (NGS). However, there is a lack of comprehensive understanding of errors introduced at various steps of a conventional NGS workflow, such as sample handling, library preparation, PCR enrichment, and sequencing. In this study, we use current NGS technology to systematically investigate these questions. RESULTS: By evaluating read-specific error distributions, we discover that the substitution error rate can be computationally suppressed to 10-5 to 10-4, which is 10- to 100-fold lower than generally considered achievable (10-3) in the current literature. We then quantify substitution errors attributable to sample handling, library preparation, enrichment PCR, and sequencing by using multiple deep sequencing datasets. We find that error rates differ by nucleotide substitution types, ranging from 10-5 for A>C/T>G, C>A/G>T, and C>G/G>C changes to 10-4 for A>G/T>C changes. Furthermore, C>T/G>A errors exhibit strong sequence context dependency, sample-specific effects dominate elevated C>A/G>T errors, and target-enrichment PCR led to ~ 6-fold increase of overall error rate. We also find that more than 70% of hotspot variants can be detected at 0.1 ~ 0.01% frequency with the current NGS technology by applying in silico error suppression. CONCLUSIONS: We present the first comprehensive analysis of sequencing error sources in conventional NGS workflows. The error profiles revealed by our study highlight new directions for further improving NGS analysis accuracy both experimentally and computationally, ultimately enhancing the precision of deep sequencing.


Assuntos
Sequenciamento de Nucleotídeos em Larga Escala/normas , Neoplasias/genética , Reação em Cadeia da Polimerase/normas , Análise de Sequência de DNA/normas , Software , Estudos de Casos e Controles , Humanos , Mutação , Controle de Qualidade
5.
Leukemia ; 33(6): 1387-1399, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-30575821

RESUMO

Acute promyelocytic leukemia (APL) is characterized by t(15;17)(q22;q21), resulting in a PML-RARA fusion that is the master driver of APL. A few cases that cannot be identified with PML-RARA by using conventional methods (karyotype analysis, FISH, and RT-PCR) involve abnormal promyelocytes that are fully in accordance with APL in morphology, cytochemistry, and immunophenotype. To explore the mechanisms involved in pathogenesis and recurrence of morphologically diagnosed APL, we performed comprehensive variant analysis by next-generation sequencing in 111 pediatric patients morphologically diagnosed as APL. Structural variant (SV) analysis in 120 DNA samples from both diagnosis and relapse stage identified 95 samples with RARA rearrangement (including 94 with PML-RARA and one with NPM-RARA) and two samples with KMT2A rearrangement. In the eligible 13 RNA samples without any RARA rearrangement at diagnosis, one case each with CPSF6-RARG, NPM1-CCDC28A, and TBC1D15-RAB21 and two cases with a TBL1XR1-RARB fusion were discovered. These uncovered fusion genes strongly suggested their contributions to leukemogenesis as driver alternations and APL phenotype may arise by abnormalities of other members of the nuclear receptor superfamily involved in retinoid signaling (RARB or RARG) or even by mechanisms distinct from the formation of aberrant retinoid receptors. Single-nucleotide variant (SNV) analysis in 77 children (80 samples) with RARA rearrangement showed recurrent alternations of primary APL in FLT3, WT1, USP9X, NRAS, and ARID1A, with a strong potential for involvement in pathogenesis, and WT1 as the only recurrently mutated gene in relapsed APL. WT1, NPM1, NRAS, FLT3, and NSD1 were identified as recurrently mutated in 17 primary samples without RARA rearrangement and WT1, NPM1, TP53, and RARA as recurrently mutated in 9 relapsed samples. The survival of APL with RARA rearrangement is much better than without RARA rearrangement. Thus, patients morphologically diagnosed as APL that cannot be identified as having a RARA rearrangement are more reasonably classified as a subclass of AML other than APL, and individualized treatment should be considered according to the genetic abnormalities.


Assuntos
Biomarcadores Tumorais/genética , Células Precursoras de Granulócitos/patologia , Leucemia Promielocítica Aguda/genética , Mutação , Recidiva Local de Neoplasia/genética , Proteínas de Fusão Oncogênica/genética , Translocação Genética , Adolescente , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Estudos de Casos e Controles , Criança , Pré-Escolar , Feminino , Seguimentos , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Células Precursoras de Granulócitos/metabolismo , Humanos , Lactente , Leucemia Promielocítica Aguda/diagnóstico , Leucemia Promielocítica Aguda/tratamento farmacológico , Masculino , Recidiva Local de Neoplasia/diagnóstico , Recidiva Local de Neoplasia/tratamento farmacológico , Prognóstico , Estudos Retrospectivos , Taxa de Sobrevida
6.
Hematol Oncol ; 36(4): 679-688, 2018 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-30133806

RESUMO

Long-term follow-up data for childhood acute lymphoblastic leukemia (ALL) are scarce in China because of lacking population-based and hospitalized registry system. This retrospective study, conducted at Shanghai's Children's Medical Center in China (SCMC), aimed to investigate the long-term results of childhood ALL and to identify prognostic factors. The Pediatric Oncology Network Database, designed by St. Jude Children's Research Hospital, USA, were used to collect data for the enrolled patients starting in 2005. From 2005 to 2014, 1085 evaluable patients with ALL aged 1 to 18 years old were enrolled and treated using SCMC-ALL-2005 risk-stratified protocol. Complete remission was achieved in 95.6% of patients. At 5 and 10 years, the event-free survival rate was 68.3 ± 1.4% and 64.6 ± 1.6%, and the overall survival rate was 80.0 ± 1.2% and 76.3 ± 1.6%, respectively. The 5-year event-free survival rates were 81.8 ± 2.0%, 67.0 ± 1.9%, and 14.3 ± 4.0% for patients in low-risk, intermediate-risk, and high-risk groups, respectively. The cumulative risk of relapse was 24.5% at 10 years. Induction failure conferred worse prognosis. Patients younger than 1 year of age at diagnosis, intermediate-risk/high-risk group, male gender, and positive minimal residual disease (MRD) results at day 55, both in the univariate and multivariate analysis, were associated with significantly worse prognosis (P < .05). Patients with positive MRD at both day 35 and day 55 were related to a significantly poor outcome (P < .0001), but not for patients with negitive MRD at day 35. The overall outcomes for ALL patients treated with protocol SCMC-ALL-2005 in SCMC are lower than in developed countries. Factors including age, gender, risk group and MRD results at day 55 were associated with treatment outcomes in childhood ALL.


Assuntos
Leucemia-Linfoma Linfoblástico de Células Precursoras/terapia , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Criança , Pré-Escolar , China/epidemiologia , Feminino , Transplante de Células-Tronco Hematopoéticas , Humanos , Lactente , Masculino , Leucemia-Linfoma Linfoblástico de Células Precursoras/sangue , Leucemia-Linfoma Linfoblástico de Células Precursoras/epidemiologia , Sistema de Registros , Estudos Retrospectivos , Resultado do Tratamento
7.
Exp Hematol ; 66: 32-41.e8, 2018 10.
Artigo em Inglês | MEDLINE | ID: mdl-30031030

RESUMO

Fanconi anemia (FA) is a rare recessive disease characterized by progressive bone marrow failure, congenital abnormalities, and increased incidence of cancers. To date, mutations in 22 genes can cause FA or an FA-like phenotype. In China, in addition to clinical information, FA diagnosis primarily relies on genetic sequencing because the chromosome breakage test is rarely performed. Here, we employed multiple genetic diagnostic tools (DNA sequencing, multiplex ligation-dependent probe amplification, and chromosome microarray) and a variant-based functional assay platform to investigate the genetic cause in 25 Chinese suspected FA patients. A total of 45 distinct candidate variants were detected in six FA genes (FA-A, FA-B, FA-C, FA-D2, FA-G, and FA-J), of which 36 were novel. Eight missense variants and one indel variant were unable to restore FANCD2 mono-ubiquitination and mitomycin C resistance in a panel of FA indicator cell lines, indicating that these mutations are deleterious. Three missense variants (FANCA-L424V, FANCC-E273K, and FANCG-A153G) were harmless. Finally, 23 patients were molecularly diagnosed with FA, consistent with their clinical phenotype. In the FA-A subgroup, large deletions accounted for 14% of the disease-causing variants. We have established a comprehensive molecular diagnostic workflow for Chinese FA patients that can substitute for standard FA cytogenetic analysis.


Assuntos
Proteína do Grupo de Complementação A da Anemia de Fanconi/genética , Proteína do Grupo de Complementação C da Anemia de Fanconi/genética , Proteína do Grupo de Complementação D2 da Anemia de Fanconi/genética , Proteína do Grupo de Complementação G da Anemia de Fanconi/genética , Anemia de Fanconi/genética , Mutação , Sequência de Bases , Criança , Pré-Escolar , China , Éxons , Anemia de Fanconi/diagnóstico , Anemia de Fanconi/etnologia , Anemia de Fanconi/patologia , Feminino , Expressão Gênica , Humanos , Lactente , Íntrons , Masculino , Reação em Cadeia da Polimerase Multiplex , Análise de Sequência de DNA , Sequenciamento Completo do Exoma
8.
Pediatr Blood Cancer ; 65(10): e27266, 2018 10.
Artigo em Inglês | MEDLINE | ID: mdl-29943896

RESUMO

BACKGROUND: Mixed-lineage leukemia (MLL) with multifarious partner genes leads to aggressive leukemia with dismal outcomes. METHODS: Using panel-based targeted sequencing, we examined 90 cases with MLL-rearranged (MLL-r) childhood acute leukemia, including 55 with acute lymphoblastic leukemia (ALL) and 35 with acute myeloid leukemia (AML). RESULTS: MLL breakpoints and complete rearrangements were identified. A total of 37.8% (34/90) of patients displayed a single direct MLL fusion gene, 15.6% (14/90) carried a single reciprocal fusion, and 27.8% (25/90) had both reciprocal MLL fusion alleles. The remaining 17 MLL-r cases exhibited complex translocations with homozygous disruptions on chromosome 11 or two breakpoints on the same MLL allele with a deletion of functional regions. A total of 77 patients (45 ALL and 32 AML) received chemotherapy with a median follow-up of 2.5 years. Unexpectedly, we identified children with reciprocal MLL fusions who exhibited relatively favorable outcomes compared with those in children with complex translocations or a single direct MLL fusion allele (66.1% vs. 24.6% and 27.6%, P = 0.001). Reciprocal MLL fusion may be functionally rescued by a partially truncated MLL protein. CONCLUSION: Comprehensive MLL-r analysis by targeted next-generation sequencing can provide detailed molecular information and is helpful for precise stratified treatment and clinical prognosis determination.


Assuntos
Histona-Lisina N-Metiltransferase/genética , Leucemia Mieloide Aguda/genética , Proteína de Leucina Linfoide-Mieloide/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , Criança , Pré-Escolar , Feminino , Rearranjo Gênico , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Lactente , Estimativa de Kaplan-Meier , Leucemia Mieloide Aguda/mortalidade , Leucemia Mieloide Aguda/patologia , Masculino , Leucemia-Linfoma Linfoblástico de Células Precursoras/mortalidade , Leucemia-Linfoma Linfoblástico de Células Precursoras/patologia , Prognóstico , Intervalo Livre de Progressão , Translocação Genética
9.
J Pediatr Hematol Oncol ; 40(5): e299-e304, 2018 07.
Artigo em Inglês | MEDLINE | ID: mdl-29702541

RESUMO

Fanconi anemia (FA) is a rare autosomal recessive or X-linked disorder with highly variable clinical manifestations and an incidence of ∼1 to 5 in 1 million births. To date, 15 bona fide FA genes have been reported to be responsible for the known FA complementation groups and the FANCA gene accounts for almost 60%. In the present study, we report a special Chinese family, which has 2 children with classic FA characteristics. Via 2-step analysis of the whole-exome sequencing data and verification using multiplex ligation-dependent probe amplification test, one child was found to have a novel compound heterozygous mutation of a splicing variant (c.1471-1G>A) and a large intragenic deletion (exons 23-30 del) of the FANCA gene. The other child had the same splicing variant and another novel large deletion (exons 1-18 del) in the FANCA gene. Clone sequencing showed the c.1471-1G>A variant generate an altered transcript with 1 cryptic splice site in intron 15, resulting in a premature termination codon (p.Val490HisfsX6). This study not only shows the complexity of FA molecular diagnosis via comprehensively studying the FA pathogenic genes and the mutational spectrum, but also has significant reference value for the future molecular diagnosis of FA.


Assuntos
Família , Proteína do Grupo de Complementação A da Anemia de Fanconi/genética , Anemia de Fanconi/diagnóstico , Anemia de Fanconi/genética , Mutação , Sítios de Splice de RNA , Grupo com Ancestrais do Continente Asiático , Criança , China , Códon de Terminação , Humanos , Masculino
10.
Proc Natl Acad Sci U S A ; 115(2): 373-378, 2018 01 09.
Artigo em Inglês | MEDLINE | ID: mdl-29279377

RESUMO

T-cell acute lymphoblastic leukemia (T-ALL) is a clonal malignancy of immature T cells. Recently, the next-generation sequencing approach has allowed systematic identification of molecular features in pediatric T-ALL. Here, by performing RNA-sequencing and other genomewide analysis, we investigated the genomic landscape in 61 adult and 69 pediatric T-ALL cases. Thirty-six distinct gene fusion transcripts were identified, with SET-NUP214 being highly related to adult cases. Among 18 previously unknown fusions, ZBTB16-ABL1, TRA-SALL2, and involvement of NKX2-1 were recurrent events. ZBTB16-ABL1 functioned as a leukemogenic driver and responded to the effect of tyrosine kinase inhibitors. Among 48 genes with mutation rates >3%, 6 were newly found in T-ALL. An aberrantly overexpressed short mRNA transcript of the SLC17A9 gene was revealed in most cases with overexpressed TAL1, which predicted a poor prognosis in the adult group. Up-regulation of HOXA, MEF2C, and LYL1 was often present in adult cases, while TAL1 overexpression was detected mainly in the pediatric group. Although most gene fusions were mutually exclusive, they coexisted with gene mutations. These genetic abnormalities were correlated with deregulated gene expression markers in three subgroups. This study may further enrich the current knowledge of T-ALL molecular pathogenesis.


Assuntos
Regulação Leucêmica da Expressão Gênica , Proteínas de Fusão Oncogênica/genética , Leucemia-Linfoma Linfoblástico de Células T Precursoras/genética , Transcriptoma , Adulto , Criança , Estudos de Coortes , Perfilação da Expressão Gênica/métodos , Ontologia Genética , Células HEK293 , Humanos , Células Jurkat , Estimativa de Kaplan-Meier , Mutação
11.
Ann Hematol ; 96(8): 1389-1397, 2017 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-28623394

RESUMO

Hematopoietic stem cell transplantation (HSCT) using an optimized conditioning regimen is essential for the long-term survival of patients with inherited bone marrow failure syndromes (IBMFS). We report HSCT in 24 children with Fanconi anemia (FA, n = 12), Diamond-Blackfan anemia (DBA, n = 7), and dyskeratosis congenita (DC, n = 5) from a single HSCT center. The graft source was peripheral blood stem cells (n = 19) or cord blood stem cells (n = 5). FA and DC patients received reduced-intensity conditioning, while DBA patients had myeloablative conditioning. The median numbers of infused mononuclear cells and CD34+ cells were 14.20 × 108/kg and 4.3 × 106/kg, respectively. The median time for neutrophil and platelet recovery was 12 and 18 days, respectively. Complete donor engraftment was achieved in 23 of 24 patients. There was one primary graft failure. During a median follow-up of 27.5 months (range, 2-130 months), the overall survival in all patients was 95.8%. The incidence of grade II-III acute graft versus host disease (GvHD) and chronic GvHD was 29.2% and 16.7%, respectively. We conclude that HSCT can be a curative option for patients with IBMFS. Modification of the conditioning regimen based on the type of disease may lead to encouraging long-term outcomes.


Assuntos
Anemia Aplástica/terapia , Doenças da Medula Óssea/terapia , Transplante de Células-Tronco de Sangue do Cordão Umbilical/métodos , Transplante de Células-Tronco Hematopoéticas/métodos , Hemoglobinúria Paroxística/terapia , Transplante de Células-Tronco de Sangue Periférico/métodos , Adolescente , Anemia de Diamond-Blackfan/terapia , Criança , Pré-Escolar , Transplante de Células-Tronco de Sangue do Cordão Umbilical/efeitos adversos , Seleção do Doador , Disceratose Congênita/terapia , Anemia de Fanconi/terapia , Feminino , Doença Enxerto-Hospedeiro/diagnóstico , Doença Enxerto-Hospedeiro/etiologia , Transplante de Células-Tronco Hematopoéticas/efeitos adversos , Hematúria/diagnóstico , Hematúria/etiologia , Humanos , Lactente , Estimativa de Kaplan-Meier , Masculino , Transplante de Células-Tronco de Sangue Periférico/efeitos adversos , Condicionamento Pré-Transplante
13.
J Cancer Res Clin Oncol ; 143(7): 1327-1335, 2017 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-28283783

RESUMO

This retrospective study evaluated the long-term outcomes and prognostic indicators of patients with stage 4 neuroblastoma who were older than 18 months at diagnosis. The medical records of 118 such children who were treated at Shanghai Children's Medical Center, China, from June 1998-December 2013 were reviewed. Event-free survival (EFS) and overall survival (OS) were analyzed by log-rank tests. Of the 118 patients, 14 improving patients did not complete treatment because of parental decisions, and 1 patient died during surgery. Of the 103 patients who completed the comprehensive protocols, 60 (58.3%) achieved very good partial remission (VGPR), 26 (25.2%) achieved partial remission (PR) after four courses of chemotherapy, and 17 (16.5%) progressed during treatment. The response to induction (including VGPR + PR) was 83.5%. After a median follow-up of 105 months (range 36-160 months), the 5- and 10-year OS were 21 and 18%, and the EFS was 19 and 13%, respectively. EFS was significantly better for patients with normal levels of urinary vanillylmandelic acid (VMA) at diagnosis, who had complete resection of the primary tumor, who were minimal residual disease- (MRD-) negative in their bone marrow after four courses of chemotherapy, and who achieved VGPR at the end of treatment (P < 0.05). The prognosis remains poor for patients with stage 4 neuroblastoma who are older than 18 months at diagnosis. Elevated VMA level, incomplete tumor resection, persistent MRD in bone marrow, and poor curative effect are associated with worse prognosis.


Assuntos
Neuroblastoma/mortalidade , Neuroblastoma/patologia , Neuroblastoma/terapia , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Criança , Pré-Escolar , China , Intervalo Livre de Doença , Feminino , Citometria de Fluxo , Humanos , Lactente , Estimativa de Kaplan-Meier , Masculino , Análise Multivariada , Neoplasia Residual/mortalidade , Neoplasia Residual/patologia , Transplante de Células-Tronco de Sangue Periférico , Prognóstico , Modelos de Riscos Proporcionais , Indução de Remissão , Estudos Retrospectivos , Fatores de Risco , Ácido Vanilmandélico/urina
14.
Nat Commun ; 8: 14385, 2017 02 09.
Artigo em Inglês | MEDLINE | ID: mdl-28181482

RESUMO

The non-coding regions of tumour cell genomes harbour a considerable fraction of total DNA sequence variation, but the functional contribution of these variants to tumorigenesis is ill-defined. Among these non-coding variants, somatic insertions are among the least well characterized due to challenges with interpreting short-read DNA sequences. Here, using a combination of Chip-seq to enrich enhancer DNA and a computational approach with multiple DNA alignment procedures, we identify enhancer-associated small insertion variants. Among the 102 tumour cell genomes we analyse, small insertions are frequently observed in enhancer DNA sequences near known oncogenes. Further study of one insertion, somatically acquired in primary leukaemia tumour genomes, reveals that it nucleates formation of an active enhancer that drives expression of the LMO2 oncogene. The approach described here to identify enhancer-associated small insertion variants provides a foundation for further study of these abnormalities across human cancers.


Assuntos
Elementos Facilitadores Genéticos , Genoma Humano , Mutagênese Insercional/genética , Oncogenes , Adolescente , Adulto , Sequência de Bases , Linhagem Celular Tumoral , Criança , Pré-Escolar , Regulação Leucêmica da Expressão Gênica , Humanos , Lactente , Leucemia-Linfoma de Células T do Adulto/genética , Reprodutibilidade dos Testes , Adulto Jovem
15.
EBioMedicine ; 8: 173-183, 2016 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-27428428

RESUMO

Genomic landscapes of 92 adult and 111 pediatric patients with B-cell acute lymphoblastic leukemia (B-ALL) were investigated using next-generation sequencing and copy number alteration analysis. Recurrent gene mutations and fusions were tested in an additional 87 adult and 93 pediatric patients. Among the 29 newly identified in-frame gene fusions, those involving MEF2D and ZNF384 were clinically relevant and were demonstrated to perturb B-cell differentiation, with EP300-ZNF384 inducing leukemia in mice. Eight gene expression subgroups associated with characteristic genetic abnormalities were identified, including leukemia with MEF2D and ZNF384 fusions in two distinct clusters. In subgroup G4 which was characterized by ERG deletion, DUX4-IGH fusion was detected in most cases. This comprehensive dataset allowed us to compare the features of molecular pathogenesis between adult and pediatric B-ALL and to identify signatures possibly related to the inferior outcome of adults to that of children. We found that, besides the known discrepancies in frequencies of prognostic markers, adult patients had more cooperative mutations and greater enrichment for alterations of epigenetic modifiers and genes linked to B-cell development, suggesting difference in the target cells of transformation between adult and pediatric patients and may explain in part the disparity in their responses to treatment.


Assuntos
Perfilação da Expressão Gênica , Regulação Leucêmica da Expressão Gênica , Genômica , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , Transcriptoma , Adolescente , Adulto , Idoso , Medula Óssea/patologia , Criança , Pré-Escolar , Análise por Conglomerados , Variações do Número de Cópias de DNA , Genômica/métodos , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Lactente , Pessoa de Meia-Idade , Mutação , Taxa de Mutação , Proteínas de Fusão Oncogênica/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras/diagnóstico , Leucemia-Linfoma Linfoblástico de Células Precursoras/mortalidade , Prognóstico , Adulto Jovem
16.
Cancer Med ; 5(2): 337-51, 2016 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-26711002

RESUMO

The t(12;21)(p13;q22) ETV6-RUNX1 gene fusion is one of the most common chromosomal translocation in childhood acute lymphoblastic leukemia (ALL). It is associated with favorable prognosis. The identification of the genomic sequence of the breakpoint flanking regions of the ETV6-RUNX1 translocation should be the best strategy to monitor minimal residual disease (MRD) in patients with ETV6-RUNX1-positive ALL. In this study, the ETV6-RUNX1 translocation was sequenced by next-generation sequencing (NGS) in 26 patients with ETV6-RUNX1-positive ALL and re-sequenced by using the Sanger method. Interestingly, the three-way translocation, including ETV6-RUNX1, was detected in five patients. Four of them relapsed during or after therapy, while 21 patients without the three-way translocation were still in remission (P < 0.0001). The three-way translocation pattern was identical between the diagnosis and relapse samples in three patients, excluding one patient (SCMC-001245). The relapse samples retained the translocation of ETV6-RUNX1 relative to the three-way translocation t(8;12;21) at diagnosis, suggesting that the three-way translocation might be an important risk factor for relapse in patients with ETV6-RUNX1-positive ALL and should be further studied.


Assuntos
Pontos de Quebra do Cromossomo , Subunidade alfa 2 de Fator de Ligação ao Core/genética , Sequenciamento de Nucleotídeos em Larga Escala , Proteínas de Fusão Oncogênica/genética , Medula Óssea/patologia , Criança , Pré-Escolar , Cromossomos Humanos Par 12 , Cromossomos Humanos Par 21 , Evolução Clonal , Feminino , Ordem dos Genes , Loci Gênicos , Humanos , Hibridização in Situ Fluorescente , Lactente , Masculino , Leucemia-Linfoma Linfoblástico de Células Precursoras/diagnóstico , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , Recidiva , Translocação Genética
17.
Zhonghua Er Ke Za Zhi ; 53(6): 453-8, 2015 Jun.
Artigo em Chinês | MEDLINE | ID: mdl-26310557

RESUMO

OBJECTIVE: The exact mechanisms of defect closure in patients with perimembranous ventricular septal defect (PMVSD) remain unknown. We hypothesized that the expression of urokinase type plasminogen activator (uPA) and plasminogen activator inhibitor-1 (PAI-1) may mediate extracellular matrix (ECM) remodeling in aneurysms. METHOD: Seven normal heart tricuspid septal leaflet and 33 aneurysms were collected in Shanghai Renji Hospital and Shanghai Children's Medical Center from January 2008 to June 2010. Immunohistochemical expression of uPA and PAI-1 in 4 normal heart valvular tissues and 15 aneurysms was detected with immunohistochemical methods. The expression of uPA and PAI-1 mRNA in 3 normal heart valvular tissues and 7 aneurysms was studied by real time fluorescent PCR; the protein expression of uPA and PAI-1 in 4 normal heart valvular tissues and 11 aneurysms was tested with Western blotting. RESULT: The surface of the aneurysms were completely covered by endothelial cells. Two types of granulation tissue, myxoid and fibrous, were associated with the aneurismal formation. uPA were recognized predominantly in valvar interstitial cells (VICs) which located mainly in regions adjacent to the endothelium and smooth muscle cells of blood vessels. PAI-1 was found in both VICs which located mainly in granulation tissue and endothelial cells. Nine aneurysms expressed a higher uPA activity than 4 normal valvular tissues ((74.6±11.8)% vs. (49.5±7.4)%; t = 3.87, P = 0.003) and six aneurysms expressed a low uPA activity ((10.3±3.1)% vs. (49.5±7.4)%; t=11.78, P=0.000) and a high PAI-1 activity ((55.2±1.7)% vs. (50.8±3.8)%; t=2.55, P=0.034) using immunohistochemical methods. uPA / PAI-1 ratio of protein expression tested by Western blot was 0.88±0.22 in four normal heart vavular tissues; five aneurysms expressed high uPA activity and low PAI-1 activity and uPA/PAI-1 ratio was 4.26±2.04; while the other 6 cases expressed low uPA activity and high PAI-1 activity and uPA/PAI-1 ratio was 0.30±0.07; the difference among the three groups was statistically significant (P<0.05). The rate of uPA/PAI-1 in relative copy of mRNA expression among normal heart valvular tissue, high uPA expressed aneurysms and low uPA expressed aneurysms are also significantly different (2.14±0.17 vs. 0.45±0.04; 2.14±0.17 vs. 4.38±1.41, P<0.05) respectively. CONCLUSION: The expression of uPA and PAI-1 in VICs suggests that interactions among these molecules contribute to the aneurysm formation and development. This provides a potential mechanism for defect closure in patients with PMVSD.


Assuntos
Aneurisma/patologia , Comunicação Interventricular/patologia , Inibidor 1 de Ativador de Plasminogênio/metabolismo , Ativador de Plasminogênio Tipo Uroquinase/metabolismo , Western Blotting , China , Células Endoteliais/citologia , Matriz Extracelular/metabolismo , Tecido de Granulação/patologia , Humanos , Imuno-Histoquímica , RNA Mensageiro
18.
Nat Med ; 21(6): 563-71, 2015 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-25962120

RESUMO

Relapse is the leading cause of mortality in children with acute lymphoblastic leukemia (ALL). Among chemotherapeutics, thiopurines are key drugs in ALL combination therapy. Using whole-exome sequencing, we identified relapse-specific mutations in the phosphoribosyl pyrophosphate synthetase 1 gene (PRPS1), which encodes a rate-limiting purine biosynthesis enzyme, in 24/358 (6.7%) relapsed childhood B cell ALL (B-ALL) cases. All individuals who harbored PRPS1 mutations relapsed early during treatment, and mutated ALL clones expanded exponentially before clinical relapse. Our functional analyses of PRPS1 mutants uncovered a new chemotherapy-resistance mechanism involving reduced feedback inhibition of de novo purine biosynthesis and competitive inhibition of thiopurine activation. Notably, the de novo purine synthesis inhibitor lometrexol effectively abrogated PRPS1 mutant-driven drug resistance. These results highlight the importance of constitutive activation of the de novo purine synthesis pathway in thiopurine resistance, and they offer therapeutic strategies for the treatment of relapsed and thiopurine-resistant ALL.


Assuntos
Retroalimentação Fisiológica/efeitos dos fármacos , Leucemia de Células B/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , Ribose-Fosfato Pirofosfoquinase/genética , Adolescente , Criança , Pré-Escolar , Exoma/genética , Feminino , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Lactente , Leucemia de Células B/tratamento farmacológico , Leucemia de Células B/patologia , Masculino , Mercaptopurina/administração & dosagem , Mutação , Leucemia-Linfoma Linfoblástico de Células Precursoras/tratamento farmacológico , Leucemia-Linfoma Linfoblástico de Células Precursoras/patologia , Purinas/biossíntese , Recidiva , Ribose-Fosfato Pirofosfoquinase/química , Tetra-Hidrofolatos/administração & dosagem
19.
Zhonghua Er Ke Za Zhi ; 53(11): 817-23, 2015 Nov.
Artigo em Chinês | MEDLINE | ID: mdl-26758319

RESUMO

OBJECTIVE: To enrich our national database with data of rare diseases by analyzing molecular diagnosis and hematopoietic stem cell transplantation (HSCT) in children with inherited bone marrow failure syndromes (IBMFS). METHOD: Next-generation sequencing (NGS)-based genetic diagnosis panel was applied for the clinical diagnosis and management of IBMFS. Retrospective analysis was performed on clinical and genetic data of 17 consecutive children who received HSCT over a long time interval (November. 2005-June 2015). RESULT: Three patients were diagnosed only by clinical manifestation before 2012. After that NGS-based genetic diagnosis panel was used to identify IBMFS-related genes in 12/14.IBMFS patients (except two Diamond-Blackfan anemia (DBA) patients). Two Fanconi anemia (FA) patients were confirmed to be new variations through family-genotype-analysis and 3 families accepted prenatal diagnosis to avoid birth of affected fetuses. Seventeen IBMFS patients (10 FA,5 DBA and 2 dyskeratosis congenital (DKC)) were treated with HSCT from matched sibling donors (n=2), matched unrelated donors (n=8) or mismatched unrelated donors (n=7). The source of stem cells for transplantation included peripheral blood (n=12) and cord blood (n=5). With regard to the conditioning regimens, FA and DKC patients received fludarabine-based reduced intensity conditioning, while DBA patients received classical busulfan-based myeloablative conditioning. Median age at the time of HSCT was 36 months (7-156 months). The number of infused mononuclear cells and CD34⁺ cells was (10.6 ± 6.7) × 108 and (5.9 ± 7.0) × 106 per kilogram of recipient body weight, respectively. The median number of days to neutrophil recovery was 13 days after HSCT (range: 10-19 days). Platelet recovery was faster in the PBSCT group than in the CBT group ((16.3 ± 6.0) days vs. (30.0 ± 17.1) days,t=-2.487,P=0.026). During a median follow-up of 17 months (range: 2-114 months), except one FA patient who was transplanted with HLA-matched unrelated cord blood (CB) died from pneumonia and heart failure because of engraftment failure, other 16 children are alive after the successful HSCT. The failure-free survival rate of the patients three years after HSCT was 94%. CONCLUSION: NGS-based molecular diagnosis technology and effective HSCT have significantly facilitated the treatment of children with IBMFS in our country, and our national database about this rare disease is to be further exploited.


Assuntos
Transplante de Células-Tronco Hematopoéticas , Hemoglobinúria Paroxística/diagnóstico , Hemoglobinúria Paroxística/terapia , Anemia Aplástica , Anemia de Diamond-Blackfan/terapia , Doenças da Medula Óssea , Criança , Disceratose Congênita/terapia , Anemia de Fanconi/terapia , Sangue Fetal , Hemoglobinúria Paroxística/genética , Humanos , Estudos Retrospectivos , Irmãos , Taxa de Sobrevida , Condicionamento Pré-Transplante , Doadores não Relacionados , Vidarabina/análogos & derivados , Vidarabina/uso terapêutico
20.
Oncotarget ; 5(21): 10732-44, 2014 Nov 15.
Artigo em Inglês | MEDLINE | ID: mdl-25313141

RESUMO

B cell acute lymphoblastic leukemia (B-ALL) is the most common hematological malignancy diagnosed in children, and blockade of the abnormally activated PI3Kδ displayed promising outcomes in B cell acute or chronic leukemias, but the mechanisms are not well understood. Here we report a novel PI3Kδ selective inhibitor X-370, which displays distinct binding mode with p110δ and blocks constitutively active or stimulus-induced PI3Kδ signaling. X-370 significantly inhibited survival of human B cell leukemia cells in vitro, with associated induction of G1 phase arrest and apoptosis. X-370 abrogated both Akt and Erk1/2 signaling via blockade of PDK1 binding to and/or phosphorylation of MEK1/2. Forced expression of a constitutively active MEK1 attenuated the antiproliferative activity of X-370. X-370 preferentially inhibited the survival of primary pediatric B-ALL cells displaying PI3Kδ-dependent Erk1/2 phosphorylation, while combined inhibition of PI3Kδ and MEK1/2 displayed enhanced activity. We conclude that PI3Kδ inhibition led to abrogation of both Akt and Erk1/2 signaling via a novel PI3K-PDK1/MEK1/2-Erk1/2 signaling cascade, which contributed to its efficacy against B-ALL. These findings support the rationale for clinical testing of PI3Kδ inhibitors in pediatric B-ALL and provide insights needed to optimize the therapeutic strategy.


Assuntos
Benzimidazóis/farmacologia , MAP Quinase Quinase 1/antagonistas & inibidores , MAP Quinase Quinase 2/antagonistas & inibidores , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Leucemia-Linfoma Linfoblástico de Células Precursoras B/tratamento farmacológico , Inibidores de Proteínas Quinases/farmacologia , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-akt/antagonistas & inibidores , Purinas/farmacologia , Apoptose/efeitos dos fármacos , Benzimidazóis/química , Western Blotting , Proliferação de Células/efeitos dos fármacos , Desenho de Drogas , Humanos , Imunoprecipitação , MAP Quinase Quinase 1/metabolismo , MAP Quinase Quinase 2/metabolismo , Estrutura Molecular , Fosfatidilinositol 3-Quinases/metabolismo , Fosforilação/efeitos dos fármacos , Leucemia-Linfoma Linfoblástico de Células Precursoras B/metabolismo , Leucemia-Linfoma Linfoblástico de Células Precursoras B/patologia , Inibidores de Proteínas Quinases/química , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Purinas/química , Transdução de Sinais/efeitos dos fármacos , Células Tumorais Cultivadas
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