RESUMEN
T-cell lymphoblastic lymphoma (T-LBL) and T-cell acute lymphoblastic leukemia (T-ALL) have common and distinguishing clinical and molecular features. Molecular prognostic factors are needed for T-LBL. We assessed the prevalence and prognostic impact of the T-cell receptor ß (TRB)::NOTCH1 fusion in 192 T-LBL and 167 pediatric T-ALL patients, using novel multiplex PCR and genomic capture high-throughput sequencing techniques. The fusion was detected in twelve T-LBL patients (6.3 %) but in none of the T-ALL patients (p=0.0006, Fisher's exact test). In T-LBL, the TRB::NOTCH1 fusion was associated with a significantly higher incidence of relapse (67% versus 17% in gene fusion-negative patients, p<0.001, Fisher's exact test). The breakpoint in TRB, was most frequently located in J2-7 (n=6). In NOTCH1, the breakpoints varied between exon 24 and 27. Consequently, a truncated NOTCH1 with its dimerization, regulation and signal transduction domains gets controlled by strong TRB enhancer elements. This study reveals a novel recurrent genetic variant with significant prognostic relevance in T-LBL, which was absent in T-ALL. The TRB::NOTCH1 fusion in T-LBL suggests a possible unique pathogenic mechanism divergent from T-ALL. Further studies will validate the role of the TRB::NOTCH1 fusion as prognostic marker in T-LBL and elucidate its pathogenic mechanisms.
RESUMEN
T-cell lymphoblastic lymphoma (T-LBL) is a heterogeneous malignancy of lymphoblasts committed to T-cell lineage. The dismal outcomes (15%-30%) after T-LBL relapse warrant establishing risk-based treatment. To our knowledge, this study presents the first comprehensive, systematic, integrated, genome-wide analysis including relapsed cases that identifies molecular markers of prognostic relevance for T-LBL. NOTCH1 was identified as the putative driver for T-LBL. An activated NOTCH/PI3K-AKT signaling axis and alterations in cell cycle regulators constitute the core oncogenic program for T-LBL. Mutated KMT2D was identified as a prognostic marker. The cumulative incidence of relapse was 47% ± 17% in patients with KMT2D mutations, compared with 14% ± 3% in wild-type KMT2D. Structural analysis of the mutated domains of KMT2D revealed a plausible impact on structure and functional consequences. These findings provide new insights into the pathogenesis of T-LBL, including high translational potential. The ongoing LBL 2018 trial (www.clinicaltrials.gov #NCT04043494) allows for prospective validation and subsequent fine tuning of the stratification criteria for T-LBL risk groups to improve survival of pediatric patients.
Asunto(s)
Biomarcadores de Tumor/genética , Proteínas de Unión al ADN/genética , Genómica/métodos , Proteínas de Neoplasias/genética , Fosfatidilinositol 3-Quinasas/genética , Leucemia-Linfoma Linfoblástico de Células T Precursoras/patología , Proteínas Proto-Oncogénicas c-akt/genética , Receptor Notch1/genética , Adolescente , Niño , Femenino , Estudios de Seguimiento , Regulación Neoplásica de la Expresión Génica , Genoma Humano , Humanos , Masculino , Leucemia-Linfoma Linfoblástico de Células T Precursoras/genética , Pronóstico , Tasa de SupervivenciaRESUMEN
Low incidence and molecular heterogeneity of pediatric T-cell lymphoblastic lymphoma (T-LBL) require an international, large-scale effort to identify novel clinical biomarkers. The ongoing international clinical trial LBL2018 (NCT04043494) represents an ideal opportunity to implement a common analytic approach. Targeted next-generation sequencing is well-suited for this purpose; however, selection of relevant target genes for T-LBL remains subject of ongoing debates. Our group has recently designed and evaluated a first target panel of 80 candidate genes for T-LBL. The present study aimed at developing a novel optimized gene panel for large-scale application and to promote an international agreement on a common core panel. Small sequence variants obtained from our former study were systematically analyzed and classified with regards to pathogenic relevance, to prioritize candidate genes. Additional genes were curated from literature and online databases for a more comprehensive analysis of relevant functions and signaling pathways. The new target panel TGP-T-LBL entails 84 candidate genes which are key actors in NOTCH, PI3K-AKT, JAK-STAT, RAS signaling, epigenetic regulation, transcription, DNA repair, cell cycle regulation, and ribosomal function. From our former gene panel, 35 out of 80 candidate genes were selected for the novel panel. Forty-six out of 84 genes are currently being analyzed in the ongoing international trial LBL2018. Exploratory analysis of prognostic relevance on mutation-level suggested a potential association of PIK3CA variants c.1624G>A(p.Glu542Lys) and c.1633G>A(p.Glu545Lys) to occurrence of relapse, emphasizing particular relevance of mutation analysis in PI3K-AKT signaling. Our approach promotes comprehensive and clinically relevant mutational profiling of pediatric T-LBL.
Asunto(s)
Linfoma de Células T , Leucemia-Linfoma Linfoblástico de Células Precursoras , Leucemia-Linfoma Linfoblástico de Células T Precursoras , Biología , Niño , ADN , Epigénesis Genética , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Linfoma de Células T/genética , Mutación , Recurrencia Local de Neoplasia/genética , Fosfatidilinositol 3-Quinasas/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , Leucemia-Linfoma Linfoblástico de Células T Precursoras/genética , Proteínas Proto-Oncogénicas c-akt/genética , Análisis de Secuencia de ADN , Linfocitos TRESUMEN
BACKGROUND: Mature aggressive B-cell lymphomas are heterogenous malignancies that make up more than half of all diagnosed non-Hodgkin lymphoma in children and adolescents. The overall survival rate increased over the last decades to 80%-90% due to fine tuning of polychemotherapy. However, new therapeutic implications are needed to further increase the overall survival. Current clinical trials analyze the therapeutic effect of rituximab in pediatric patients, while the mechanism of action in vivo is still not fully understood. METHODS: Effector molecules important for tumor defense were analyzed before and at day 5 after rituximab treatment via flow cytometry. Serum rituximab levels were measured with an ELISA. RESULTS: We evaluated patient parameters that may affect treatment response in relation to rituximab administration and serum rituximab levels. We indeed found a reduction of Fcγ receptor (FcγR) II levels after rituximab treatment in monocyte subtypes, whereas FcγRI expression was significantly increased. Serum levels of proinflammatory marker proteins S100A8/A9 and S100A12 significantly decreased after treatment to normal levels from an overall proinflammatory state before treatment. CD57, perforin, and granzyme B expression decreased after treatment, comprising a less cytolytic natural killer (NK) cell population. CONCLUSION: The highlighted effects of rituximab treatment on patient's immune response help in understanding the biology behind tumor defense mechanisms and effector function. After subsequent studies, these novel insights might be translated into patient care and could contribute to improve treatment of pediatric patients with mature aggressive B-cell lymphoma.
Asunto(s)
Linfoma de Células B , Linfoma no Hodgkin , Adolescente , Niño , Humanos , Células Asesinas Naturales , Linfoma de Células B/tratamiento farmacológico , Linfoma no Hodgkin/tratamiento farmacológico , Receptores de IgG , Rituximab/uso terapéuticoRESUMEN
Influenza A virus (IAV) defective RNAs are generated as byproducts of error-prone viral RNA replication. They are commonly derived from the larger segments of the viral genome and harbor deletions of various sizes resulting in the generation of replication incompatible viral particles. Furthermore, small subgenomic RNAs are known to be strong inducers of pattern recognition receptor RIG-I-dependent type I interferon (IFN) responses. The present study identifies a novel IAV-induced defective RNA derived from the PB2 segment of A/Thailand/1(KAN-1)/2004 (H5N1). It encodes a 10 kDa protein (PB2∆) sharing the N-terminal amino acid sequence of the parental PB2 protein followed by frame shift after internal deletion. PB2∆ induces the expression of IFNß and IFN-stimulated genes by direct interaction with the cellular adapter protein MAVS, thereby reducing viral replication of IFN-sensitive viruses such as IAV or vesicular stomatitis virus. This induction of IFN is completely independent of the defective RNA itself that usually serves as pathogen-associated pattern and thus does not require the cytoplasmic sensor RIG-I. These data suggest that not only defective RNAs, but also some defective RNA-encoded proteins can act immunostimulatory. In this particular case, the KAN-1-induced defective RNA-encoded protein PB2∆ enhances the overwhelming immune response characteristic for highly pathogenic H5N1 viruses, leading to a more severe phenotype in vivo.
Asunto(s)
Virus de la Influenza A/fisiología , Interferón Tipo I/metabolismo , Infecciones por Orthomyxoviridae/metabolismo , ARN Viral/genética , ARN Polimerasa Dependiente del ARN/metabolismo , Proteínas Virales/metabolismo , Animales , Northern Blotting , Western Blotting , Pruebas de Hemaglutinación , Inmunoprecipitación , Interferón Tipo I/genética , Ratones , Ratones Endogámicos BALB C , Infecciones por Orthomyxoviridae/virología , ARN Mensajero/genética , ARN Polimerasa Dependiente del ARN/genética , Reacción en Cadena en Tiempo Real de la Polimerasa , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , Células Tumorales Cultivadas , Proteínas Virales/genética , Replicación ViralRESUMEN
New deep RNA sequencing methodologies in transcriptome analyses identified a wealth of novel nonprotein-coding RNAs (npcRNAs). Recently, deep sequencing was used to delineate the small npcRNA transcriptome of the human pathogen Vibrio cholerae and 627 novel npcRNA candidates were identified. Here, we report the detection of 223 npcRNA candidates in V. cholerae by different cDNA library construction and conventional sequencing methods. Remarkably, only 39 of the candidates were common to both surveys. We therefore examined possible biasing influences in the transcriptome analyses. Key steps, including tailing and adapter ligations for generating cDNA, contribute qualitatively and quantitatively to the discrepancies between data sets. In addition, the state of 5'-end phosphorylation influences the efficiency of adapter ligation and C-tailing at the 3'-end of the RNA. Finally, our data indicate that the inclusion of sample-specific molecular identifier sequences during ligation steps also leads to biases in cDNA representation. In summary, even deep sequencing is unlikely to identify all RNA species, and caution should be used for meta-analyses among alternatively generated data sets.
Asunto(s)
Perfilación de la Expresión Génica , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Análisis de Secuencia de ARN/métodos , Vibrio cholerae/genética , Clonación Molecular/métodos , Análisis por Conglomerados , ADN Ligasas/metabolismo , Perfilación de la Expresión Génica/métodos , Secuenciación de Nucleótidos de Alto Rendimiento/normas , Modelos Biológicos , ARN Bacteriano/análisis , ARN Bacteriano/genética , ARN no Traducido/análisis , ARN no Traducido/genética , Análisis de Secuencia de ARN/normas , Estudios de Validación como Asunto , Vibrio cholerae/metabolismoRESUMEN
We experimentally identified and characterized 97 novel, non-protein-coding RNA candidates (npcRNAs) from the human pathogen Salmonella enterica serovar Typhi (hereafter referred to as S. typhi). Three were specific to S. typhi, 22 were restricted to Salmonella species and 33 were differentially expressed during S. typhi growth. We also identified Salmonella Pathogenicity Island-derived npcRNAs that might be involved in regulatory mechanisms of virulence, antibiotic resistance and pathogenic specificity of S. typhi. An in-depth characterization of S. typhi StyR-3 npcRNA showed that it specifically interacts with RamR, the transcriptional repressor of the ramA gene, which is involved in the multidrug resistance (MDR) of Salmonella. StyR-3 interfered with RamR-DNA binding activity and thus potentially plays a role in regulating ramA gene expression, resulting in the MDR phenotype. Our study also revealed a large number of cis-encoded antisense npcRNA candidates, supporting previous observations of global sense-antisense regulatory networks in bacteria. Finally, at least six of the npcRNA candidates interacted with the S. typhi Hfq protein, supporting an important role of Hfq in npcRNA networks. This study points to novel functional npcRNA candidates potentially involved in various regulatory roles including the pathogenicity of S. typhi.
Asunto(s)
ARN Bacteriano/metabolismo , ARN no Traducido/metabolismo , Salmonella typhi/genética , ADN Intergénico/química , Biblioteca de Genes , Islas Genómicas , Sistemas de Lectura Abierta , Operón , ARN sin Sentido/genética , ARN Bacteriano/genética , ARN no Traducido/genética , Salmonella typhi/metabolismo , Salmonella typhi/patogenicidadRESUMEN
Nonprotein-coding RNAs (npcRNAs) represent an important class of regulatory molecules that act in many cellular pathways. Here, we describe the experimental identification and validation of the small npcRNA transcriptome of the human malaria parasite Plasmodium falciparum. We identified 630 novel npcRNA candidates. Based on sequence and structural motifs, 43 of them belong to the C/D and H/ACA-box subclasses of small nucleolar RNAs (snoRNAs) and small Cajal body-specific RNAs (scaRNAs). We further observed the exonization of a functional H/ACA snoRNA gene, which might contribute to the regulation of ribosomal protein L7a gene expression. Some of the small npcRNA candidates are from telomeric and subtelomeric repetitive regions, suggesting their potential involvement in maintaining telomeric integrity and subtelomeric gene silencing. We also detected 328 cis-encoded antisense npcRNAs (asRNAs) complementary to P. falciparum protein-coding genes of a wide range of biochemical pathways, including determinants of virulence and pathology. All cis-encoded asRNA genes tested exhibit lifecycle-specific expression profiles. For all but one of the respective sense-antisense pairs, we deduced concordant patterns of expression. Our findings have important implications for a better understanding of gene regulatory mechanisms in P. falciparum, revealing an extended and sophisticated npcRNA network that may control the expression of housekeeping genes and virulence factors.
Asunto(s)
Plasmodium falciparum/genética , ARN no Traducido/genética , Animales , Secuencia de Bases , Exones , Perfilación de la Expresión Génica , Biblioteca de Genes , Datos de Secuencia Molecular , Plasmodium falciparum/metabolismo , ARN/genética , ARN/metabolismo , ARN sin Sentido/genética , ARN sin Sentido/metabolismo , ARN Mitocondrial , ARN Ribosómico/genética , ARN Ribosómico/metabolismo , ARN Nucleolar Pequeño/genética , ARN Nucleolar Pequeño/metabolismo , ARN no Traducido/clasificación , ARN no Traducido/metabolismo , Telómero/químicaRESUMEN
Cutaneous leishmaniasis (CL) has been reported from 20 of 31 provinces of Iran. It is a polymorphic disease, which may show various clinical manifestations. Although genetic diversity of the parasite is suggested to be one of the factors influencing the clinical manifestations in CL, there is still no data regarding the genetic variation of Leishmania major based on the sequencing of kDNA .The amplification of the kinetoplast DNA based on 21 isolates of L. major from different clinical forms of CL was carried out using nested-PCR and subsequent sequencing. The clinical presentation was basically of two types: (a) typical lesions and (b) atypical, including erythematous volcanic ulcer, multi infections, lupoid, diffuse, eczematous, verrucous, dry, and nodular lesions. Sequence analysis of the amplified kDNA was used to investigate the genetic variations among L. major isolates and correlate the findings with clinical and histological features. Leishmanial DNA was detected in 98 of 100 cases. L. major and Leishmaniatropica were detected in 97 cases and 1 case, respectively. The Sequence analysis of kDNA from 21 L. major strains showed a high genetic polymorphism of L. major causing CL in southern Iran and correlations among the geographical origin and the clinical manifestations of the disease.