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Given the poor outcome of refractory and relapsing T-ALL, identifying prognostic markers is still challenging. Using SNP-array analysis, we provide a comprehensive analysis of genomic imbalances in a cohort of 317 newly-diagnosed T-ALL patients including 135 children and 182 adults with respect to clinical and biological features and outcomes. SNP-array results identified at least one somatic genomic imbalance in virtually all T-ALL patients (~96%). Del(9)(p21) (~70%) and UPD(9)p21)/CDKN2A/B (~28%) were the most frequent genomic imbalances. Unexpectedly del(13q14)/RB1/DLEU1 (~14%) was the second more frequent CNV followed by del(6)(q15)/CASP8AP2 (~11%), del(1)(p33)/SIL-TAL1 (~11%), del(12)(p13)ETV6/CDKN1B (~9%), del(18)(p11)/PTPN2 (~9%), del(1)(p36)/RPL22 (~9%), and del(17)(q11)/NF1/SUZ12 (~8%). SNP-array also revealed distinct profiles of genomic imbalances according to age, immunophenotype, and oncogenetic subgroups. In particular, adult T-ALL patients demonstrated a significantly higher incidence of del(1)(p36)/RPL22, and del(13)(q14)/RB1/DLEU1, and lower incidence of del(9)(p21) and UPD(9p21)/CDKN2A/B. We determined a threshold of 15 genomic imbalances to stratify patients into high- and low-risk groups of relapse. Survival analysis also revealed the poor outcome, despite the low number of affected cases, conferred by the presence of chromothripsis (n=6, ~2%), del(16)(p13)/CREBBP (n=15, ~5%) as well as the newly identified recurrent gain at 6q27 involving MLLT4 (n=10, ~3%). Genomic complexity, del(16)(p13)/CREBBP and gain at 6q27 involving MLLT4 maintained their significance in multivariate analysis for survival outcome. Our study thus demonstrated that whole genome analysis of imbalances provides new insights to refine risk stratification in T-ALL.
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We previously reported a better outcome in adult and pediatric T-cell acute lymphoblastic leukemia (T-ALL) harboring NOTCH1 and/or FBXW7 mutations without alterations of K-N-RAS and PTEN genes. Availability of high-throughput next-generation sequencing strategies (NGS) led us to refine the outcome prediction in T-ALL. Targeted whole-exome sequencing of 72 T-ALL related oncogenes was performed in 198 adult T-ALLs in first remission (CR1) from the GRAALL-2003/2005 protocols (ClinicalTrial.gov, NCT00222027, NCT00327678) and 242 pediatric T-ALLs from the FRALLE2000T. This approach enabled the identification of the first NGS-based classifier in T-ALL categorizing low-risk patients as those with N/F, PHF6, or EP300 mutations, excluding N-K-RAS, PI3K pathway (PTEN, PIK3CA, and PIK3R1), TP53, DNMT3A, IDH1/2, and IKZF1 alterations, with a 5-year cumulative incidence of relapse (CIR) estimated at 21%. Conversely, the remaining patients were classified as high-risk, exhibiting a 5-year CIR estimated at 47%. We externally validated this stratification in the pediatric cohort. NGS-based classifier was highly prognostic, independently of minimal residual disease (MRD) and white blood cells counts (WBC), in both adult and pediatric cohorts. Integration of the NGS-based classifier into a comprehensive risk stratification model, including WBC count at diagnosis and MRD at the end of induction, enabled the identification of an adverse risk subgroup (25%) with a 5-year CIR estimated at 51%, and a favorable risk group (32%) with a 5-year CIR estimated at 12%. NGS-based stratification combined with WBC and MRD sharpens the prognostic classification in T-ALL and identifies a new subgroup of patients who may benefit from innovative therapeutic approaches.
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Analysis and interpretation of Ig and TCR gene rearrangements in the conventional, low-throughput way have their limitations in terms of resolution, coverage, and biases. With the advent of high-throughput, next-generation sequencing (NGS) technologies, a deeper analysis of Ig and/or TCR (IG/TR) gene rearrangements is now within reach, which impacts on all main applications of IG/TR immunogenetic analysis. To bridge the generation gap from low- to high-throughput analysis, the EuroClonality-NGS Consortium has been formed, with the main objectives to develop, standardize, and validate the entire workflow of IG/TR NGS assays for 1) clonality assessment, 2) minimal residual disease detection, and 3) repertoire analysis. This concerns the preanalytical (sample preparation, target choice), analytical (amplification, NGS), and postanalytical (immunoinformatics) phases. Here we critically discuss pitfalls and challenges of IG/TR NGS methodology and its applications in hemato-oncology and immunology.
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Hematología/métodos , Secuenciación de Nucleótidos de Alto Rendimiento , Inmunogenética/métodos , Técnicas Inmunológicas , Alelos , Biología Computacional/métodos , Reordenamiento Génico , Genes de Inmunoglobulinas , Genes Codificadores de los Receptores de Linfocitos T/genética , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Humanos , Inmunogenética/normasRESUMEN
To model the developmental pattern of human prothymocytes and thymopoiesis, we used NOD-scid/γc(-/-) mice grafted with human umbilical cord blood CD34(+) hematopoietic progenitor cells (HPCs). Human prothymocytes developed in the murine bone marrow (BM) from multipotent CD34(++)CD38(lo)lineage(-) HPCs to CD34(++)CD7(+)CD2(-) pro-T1 cells that progressed in a Notch-dependent manner to CD34(+)CD7(++)CD2(+) pro-T2 cells, which migrated to the thymus. BM prothymocyte numbers peaked 1 mo after graft, dropped at mo 2, and persisted at low levels thereafter, with only a few CD34(+)CD7(lo) prothymocytes with limited T potential being detected by mo 5. As a consequence, thymopoiesis in this xenogeneic setting began by weeks 4-6, peaked at mo 3, and decreased thenceforth. Analyzing mice grafted at 2, 4 or 8, mo of age showed that in an "older" BM, prothymocyte differentiation was perturbed and resulted in CD34(+)CD7(lo) prothymocytes with limited T potential. Whereas the early drop in BM thymopoietic activity was related to a Notch-independent loss of T potential by CD34(++)CD38(lo)lineage(-) HPCs, the later age-dependent production decline of prothymocytes was linked to a more complex mix of cell-intrinsic and microenvironmental defects. Accordingly, and contrasting with what was observed with umbilical cord blood HPCs, CD34(+) HPCs from human adult BM displayed only marginal thymopoietic activity when grafted into young 2-mo-old NOD-scid/γc(-/-) mice. These data demonstrate that the developmental pattern of BM prothymocytes during human late fetal and early postnatal life can be reproduced in humanized mice, and they suggest that onset of human thymus involution relates to decreased colonization by prothymocytes.
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Diferenciación Celular/inmunología , Células Progenitoras Linfoides/citología , Linfopoyesis/fisiología , Linfocitos T/citología , Timo/citología , Animales , Células de la Médula Ósea/citología , Linaje de la Célula/inmunología , Trasplante de Células Madre Hematopoyéticas , Humanos , Ratones , Ratones Endogámicos NOD , Ratones SCID , Análisis de Secuencia por Matrices de Oligonucleótidos , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Trasplante HeterólogoRESUMEN
Cumulative evidence indicates that MYC, one of the major downstream effectors of NOTCH1, is a critical component of T-cell acute lymphoblastic leukemia (T-ALL) oncogenesis and a potential candidate for targeted therapy. However, MYC is a complex oncogene, involving both fine protein dosage and cell-context dependency, and detailed understanding of MYC-mediated oncogenesis in T-ALL is still lacking. To better understand how MYC is interspersed in the complex T-ALL oncogenic networks, we performed a thorough molecular and biochemical analysis of MYC activation in a comprehensive collection of primary adult and pediatric patient samples. We find that MYC expression is highly variable, and that high MYC expression levels can be generated in a large number of cases in absence of NOTCH1/FBXW7 mutations, suggesting the occurrence of multiple activation pathways in addition to NOTCH1. Furthermore, we show that posttranscriptional deregulation of MYC constitutes a major alternative pathway of MYC activation in T-ALL, operating partly via the PI3K/AKT axis through down-regulation of PTEN, and that NOTCH1(m) might play a dual transcriptional and posttranscriptional role in this process. Altogether, our data lend further support to the significance of therapeutic targeting of MYC and/or the PTEN/AKT pathways, both in GSI-resistant and identified NOTCH1-independent/MYC-mediated T-ALL patients.
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Genes myc , Fosfohidrolasa PTEN/fisiología , Leucemia-Linfoma Linfoblástico de Células T Precursoras/genética , Adulto , Células Cultivadas , Niño , Regulación Leucémica de la Expresión Génica , Humanos , Células Jurkat , Fosfohidrolasa PTEN/genética , Fosfohidrolasa PTEN/metabolismo , Leucemia-Linfoma Linfoblástico de Células T Precursoras/metabolismo , Leucemia-Linfoma Linfoblástico de Células T Precursoras/patología , Procesamiento Postranscripcional del ARN/genética , Procesamiento Postranscripcional del ARN/fisiología , Transducción de Señal/genética , Activación Transcripcional/genética , TransfecciónRESUMEN
The developmental cartography of human lymphopoiesis remains incompletely understood. Here, we establish a multimodal map demonstrating that lymphoid specification follows independent direct or stepwise hierarchic routes converging toward the emergence of newly characterized CD117lo multi-lymphoid progenitors (MLPs) that undergo a proliferation arrest before entering the CD127- (NK/ILC/T) or CD127+ (B) lymphoid pathways. While the differentiation of CD127- early lymphoid progenitors is mainly driven by Flt3 signaling, emergence of their CD127+ counterparts is regulated cell-intrinsically and depends exclusively on the divisional history of their upstream precursors, including hematopoietic stem cells. Further, transcriptional mapping of differentiation trajectories reveals that whereas myeloid granulomonocytic lineages follow continuous differentiation pathways, lymphoid trajectories are intrinsically discontinuous and characterized by sequential waves of cell proliferation allowing pre-commitment amplification of lymphoid progenitor pools. Besides identifying new lymphoid specification pathways and regulatory checkpoints, our results demonstrate that NK/ILC/T and B lineages are under fundamentally distinct modes of regulation. (149 words).
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Changes in lymphocyte production patterns occurring across human ontogeny remain poorly defined. In this study, we demonstrate that human lymphopoiesis is supported by three waves of embryonic, fetal, and postnatal multi-lymphoid progenitors (MLPs) differing in CD7 and CD10 expression and their output of CD127-/+ early lymphoid progenitors (ELPs). In addition, our results reveal that, like the fetal-to-adult switch in erythropoiesis, transition to postnatal life coincides with a shift from multilineage to B lineage-biased lymphopoiesis and an increase in production of CD127+ ELPs, which persists until puberty. A further developmental transition is observed in elderly individuals whereby B cell differentiation bypasses the CD127+ compartment and branches directly from CD10+ MLPs. Functional analyses indicate that these changes are determined at the level of hematopoietic stem cells. These findings provide insights for understanding identity and function of human MLPs and the establishment and maintenance of adaptative immunity.
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Células Madre Hematopoyéticas , Linfopoyesis , Adulto , Humanos , Anciano , Diferenciación Celular , Linaje de la Célula , HematopoyesisRESUMEN
Current diagnostic standards for lymphoproliferative disorders include multiple tests for detection of clonal immunoglobulin (IG) and/or T-cell receptor (TCR) rearrangements, translocations, copy-number alterations (CNAs), and somatic mutations. The EuroClonality-NGS DNA Capture (EuroClonality-NDC) assay was designed as an integrated tool to characterize these alterations by capturing IGH switch regions along with variable, diversity, and joining genes of all IG and TCR loci in addition to clinically relevant genes for CNA and mutation analysis. Diagnostic performance against standard-of-care clinical testing was assessed in a cohort of 280 B- and T-cell malignancies from 10 European laboratories, including 88 formalin-fixed paraffin-embedded samples and 21 reactive lesions. DNA samples were subjected to the EuroClonality-NDC protocol in 7 EuroClonality-NGS laboratories and analyzed using a bespoke bioinformatic pipeline. The EuroClonality-NDC assay detected B-cell clonality in 191 (97%) of 197 B-cell malignancies and T-cell clonality in 71 (97%) of 73 T-cell malignancies. Limit of detection (LOD) for IG/TCR rearrangements was established at 5% using cell line blends. Chromosomal translocations were detected in 145 (95%) of 152 cases known to be positive. CNAs were validated for immunogenetic and oncogenetic regions, highlighting their novel role in confirming clonality in somatically hypermutated cases. Single-nucleotide variant LOD was determined as 4% allele frequency, and an orthogonal validation using 32 samples resulted in 98% concordance. The EuroClonality-NDC assay is a robust tool providing a single end-to-end workflow for simultaneous detection of B- and T-cell clonality, translocations, CNAs, and sequence variants.
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Reordenamiento Génico , Trastornos Linfoproliferativos , ADN , Genómica , Humanos , Inmunoglobulinas , Trastornos Linfoproliferativos/diagnóstico , Trastornos Linfoproliferativos/genéticaRESUMEN
The majority of haematological cancers involve the lymphoid system. They include acute lymphoblastic leukemias (ALL), which are arrested at variable stages of development and present with blood and bone marrow involvement and chronic leukemias, lymphomas and myelomas, which present with infiltration of a large variety of hematopoietic and non hematopoietic tissues by mature lymphoid cells which express a surface antigen receptor. The majority involve the B-cell lineage and the vast majority have undergone clonal rearrangement of their Ig and/or TCR rearrangements. Analysis of Ig/TCR genomic V(D)J repertoires by PCR based lymphoid clonality analysis within a diagnostic setting allows distinction of clonal from reactive lymphoproliferative disorders, clonal tracking for evidence of tumor dissemination and follow-up, identification of a lymphoid origin in undiagnosed tumors and evaluation of clonal evolution. Ig/TCR VDJ errors are also at the origin of recombinase mediated deregulated expression of a variety of proto-oncogenes in ALL, whereas in lymphoma it is increasingly clear that IgH containing translocations result from abnormalities other than VDJ errors (somatic hypermutation and/or isotype switching). In addition to this mechanistic contribution to lymphoid oncogenesis, it is possible that failure to successfully complete expression of an appropriate Ig or TCR may lead to maturation arrest in a lymphoid precursor, which may in itself contribute to altered tissue homeostasis, particularly if the arrest occurs at a stage of cellular expansion.
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Reordenamiento Génico de Linfocito B , Reordenamiento Génico de Linfocito T , Neoplasias Hematológicas , Trastornos Linfoproliferativos , Recombinación Genética , Genes de Inmunoglobulinas , Genes Codificadores de los Receptores de Linfocitos T , Neoplasias Hematológicas/genética , Neoplasias Hematológicas/inmunología , Humanos , Fenómenos del Sistema Inmunológico , Inmunoglobulinas/genética , Inmunoglobulinas/inmunología , Trastornos Linfoproliferativos/genética , Trastornos Linfoproliferativos/inmunología , Receptores de Antígenos de Linfocitos T/genética , Receptores de Antígenos de Linfocitos T/inmunología , VDJ Recombinasas/metabolismoRESUMEN
T-cell Receptor Gamma (TRG) rearrangements are commonly used to detect clonal lymphoproliferations in hematopathology, since they are rearranged in virtually all T lymphocytes and have a relatively limited recombinatorial repertoire, which reduces the risk of false negative results, at the cost of potential false positivity. We developed an initial one-tube, 2-fluorochrome EuroClonality TRG PCR multiplex (TRG-1T-2F) which was compared to the original 2-tube, 2-fluorochrome EuroClonality/BIOMED-2 TRG PCR (TRG-2T-2F) and a commercial Invivoscribe one-tube, one-fluorochrome kit (IVS-1T-1F) on a series of 239 samples, including both T-cell malignancies and reactive cases. This initial assay yielded discrepant results between the 10 participating EuroClonality laboratories when using 2 fluorochromes, leading to adoption of a final single color EuroClonality strategy (TRG-1T-1F). Compared to TRG-2T-2F, both TRG-1T-1F and IVS-1T-1F demonstrated easier interpretation and a lower risk of false positive from minor peaks in dispersed repertoires. Both generate smaller fragments and as such are likely to be better adapted to analysis of formalin-fixed paraffin-embedded (FFPE) tissue samples. Their differential performance was mainly explained by (i) superposition of biallelic rearrangements with IVS-1T-1F, due to more extensive overlapping of the repertoires and (ii) intentional omission of the TRGJP primer in TRG-1T-1F, in order to avoid the potential risk of confusion of consensus TRG V9-JP normal rearrangements with a pathological clone.
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Assessment of clonality, marker identification and measurement of minimal residual disease (MRD) of immunoglobulin (IG) and T cell receptor (TR) gene rearrangements in lymphoid neoplasms using next-generation sequencing (NGS) is currently under intensive development for use in clinical diagnostics. So far, however, there is a lack of suitable quality control (QC) options with regard to standardisation and quality metrics to ensure robust clinical application of such approaches. The EuroClonality-NGS Working Group has therefore established two types of QCs to accompany the NGS-based IG/TR assays. First, a central polytarget QC (cPT-QC) is used to monitor the primer performance of each of the EuroClonality multiplex NGS assays; second, a standardised human cell line-based DNA control is spiked into each patient DNA sample to work as a central in-tube QC and calibrator for MRD quantification (cIT-QC). Having integrated those two reference standards in the ARResT/Interrogate bioinformatic platform, EuroClonality-NGS provides a complete protocol for standardised IG/TR gene rearrangement analysis by NGS with high reproducibility, accuracy and precision for valid marker identification and quantification in diagnostics of lymphoid malignancies.
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Marcadores Genéticos/genética , Inmunoglobulinas/genética , Receptores de Antígenos de Linfocitos T/genética , Biología Computacional/métodos , Reordenamiento Génico/genética , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Humanos , Neoplasia Residual/genética , Control de Calidad , Reproducibilidad de los ResultadosRESUMEN
Amplicon-based next-generation sequencing (NGS) of immunoglobulin (IG) and T-cell receptor (TR) gene rearrangements for clonality assessment, marker identification and quantification of minimal residual disease (MRD) in lymphoid neoplasms has been the focus of intense research, development and application. However, standardization and validation in a scientifically controlled multicentre setting is still lacking. Therefore, IG/TR assay development and design, including bioinformatics, was performed within the EuroClonality-NGS working group and validated for MRD marker identification in acute lymphoblastic leukaemia (ALL). Five EuroMRD ALL reference laboratories performed IG/TR NGS in 50 diagnostic ALL samples, and compared results with those generated through routine IG/TR Sanger sequencing. A central polytarget quality control (cPT-QC) was used to monitor primer performance, and a central in-tube quality control (cIT-QC) was spiked into each sample as a library-specific quality control and calibrator. NGS identified 259 (average 5.2/sample, range 0-14) clonal sequences vs. Sanger-sequencing 248 (average 5.0/sample, range 0-14). NGS primers covered possible IG/TR rearrangement types more completely compared with local multiplex PCR sets and enabled sequencing of bi-allelic rearrangements and weak PCR products. The cPT-QC showed high reproducibility across all laboratories. These validated and reproducible quality-controlled EuroClonality-NGS assays can be used for standardized NGS-based identification of IG/TR markers in lymphoid malignancies.
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Reordenamiento Génico de Linfocito T/genética , Genes Codificadores de los Receptores de Linfocitos T/genética , Marcadores Genéticos/genética , Inmunoglobulinas/genética , Neoplasia Residual/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , Biología Computacional/métodos , Genes de Inmunoglobulinas/genética , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Humanos , Receptores de Antígenos de Linfocitos T/genética , Recombinación Genética/genética , Estándares de Referencia , Reproducibilidad de los ResultadosRESUMEN
In acute myeloid leukemia (AML), coexpression of death receptors and ligands of the tumor necrosis factor (TNF) receptor/TNF-alpha superfamily on leukemic cells after chemotherapy is not always accompanied by apoptosis, suggesting that the apoptotic death receptor signaling pathway is disrupted. Because Fas-associated protein with death domain (FADD) is the main adaptor for transmitting the Fas, TNF-related apoptosis-inducing ligand receptors, and TNF receptor 1 death signal, expression of FADD was analyzed by Western blot and immunocytochemistry in leukemic cells of 70 de novo AML patients treated with the European Organization of Research and Treatment of Cancer AML-10 randomized trial before initiation of induction chemotherapy. Thirty seven percent of patients (17 of 46) with FADD negative/low (FADD(-/low)) leukemic cells had a primary refractory disease compared with 12% of FADD(+) patients (3 of 24; P = 0.05). FADD(-/low) expression was significantly associated with a worse event-free survival [EFS (P = 0.04)] and overall survival (P = 0.04). In multivariate analysis, FADD(-/low) protein expression was independently associated with a poor EFS and overall survival (P = 0.002 and P = 0.026, respectively). Importantly, FADD(-/low) protein expression predicted poor EFS even in patients with standard- or good-risk AML (P = 0.009). Thus, we identified low or absent expression of the FADD protein in leukemic cells at diagnosis as a poor independent prognostic factor that can predict worse clinical outcome even for patients with standard- or good-risk AML.
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Proteínas Adaptadoras Transductoras de Señales/análisis , Leucemia Mieloide Aguda/tratamiento farmacológico , Leucemia Mieloide Aguda/metabolismo , Adolescente , Adulto , Western Blotting , Caspasas/fisiología , Línea Celular Tumoral , Resistencia a Antineoplásicos , Proteína de Dominio de Muerte Asociada a Fas , Humanos , Inmunohistoquímica , Leucemia Mieloide Aguda/mortalidad , Microscopía Confocal , Microscopía Fluorescente , Persona de Mediana Edad , Pronóstico , Receptor fas/análisisRESUMEN
Both B and T lymphocytes have signature traits that set them apart from other cell types. They actively and repeatedly rearrange their DNA in order to produce a unique and functional antigen receptor, they have potential for massive clonal expansion upon encountering antigen via this receptor or its precursor, and they have the capacity to be extremely long lived as 'memory' cells. All three of these traits are fundamental to their ability to function as the adaptive immune response to infectious agents, but concurrently render these cells vulnerable to transformation. Thus, it is classically considered that lymphomas arise at a relatively late stage in a lymphocyte's development during the process of modifying diversity within antigen receptors, and when the cell is capable of responding to stimulus via its receptor. Attempts to understand the aetiology of lymphoma have reinforced this notion, as the most notable advances to date have shown chronic stimulation of the antigen receptor by infectious agents or self-antigens to be key drivers of these diseases. Despite this, there is still uncertainty about the cell of origin in some lymphomas, and increasing evidence that a subset arises in a more immature cell. Specifically, a recent study indicates that T-cell lymphoma, in particular nucleophosmin-anaplastic lymphoma kinase-driven anaplastic large cell lymphoma, may originate in T-cell progenitors in the thymus.
RESUMEN
T-cell acute lymphoblastic leukaemias (T-ALL) are aggressive malignant proliferations characterized by high relapse rates and great genetic heterogeneity. TAL1 is amongst the most frequently deregulated oncogenes. Yet, over half of the TAL1(+) cases lack TAL1 lesions, suggesting unrecognized (epi)genetic deregulation mechanisms. Here we show that TAL1 is normally silenced in the T-cell lineage, and that the polycomb H3K27me3-repressive mark is focally diminished in TAL1(+) T-ALLs. Sequencing reveals that >20% of monoallelic TAL1(+) patients without previously known alterations display microinsertions or RAG1/2-mediated episomal reintegration in a single site 5' to TAL1. Using 'allelic-ChIP' and CrispR assays, we demonstrate that such insertions induce a selective switch from H3K27me3 to H3K27ac at the inserted but not the germline allele. We also show that, despite a considerable mechanistic diversity, the mode of oncogenic TAL1 activation, rather than expression levels, impact on clinical outcome. Altogether, these studies establish site-specific epigenetic desilencing as a mechanism of oncogenic activation.
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Alelos , Regulación Leucémica de la Expresión Génica , Proteínas del Grupo Polycomb/genética , Leucemia-Linfoma Linfoblástico de Células T Precursoras/genética , Acetilación , Adulto , Secuencia de Bases , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Inmunoprecipitación de Cromatina , Proteínas de Unión al ADN/metabolismo , Epigénesis Genética , Sitios Genéticos , Histonas/metabolismo , Proteínas de Homeodominio/metabolismo , Humanos , Células Jurkat , Metilación , Datos de Secuencia Molecular , Mutagénesis Insercional , Proteínas Nucleares/metabolismo , Plásmidos/genética , Proteínas del Grupo Polycomb/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Análisis de Supervivencia , Proteína 1 de la Leucemia Linfocítica T Aguda , Resultado del TratamientoRESUMEN
Acute lymphoblastic leukemias (ALLs) are characterized by multistep oncogenic processes leading to cell-differentiation arrest and proliferation. Specific abrogation of maturation blockage constitutes a promising therapeutic option in cancer, which requires precise understanding of the underlying molecular mechanisms. We show that the cortical thymic maturation arrest in T-lineage ALLs that overexpress TLX1 or TLX3 is due to binding of TLX1/TLX3 to ETS1, leading to repression of T cell receptor (TCR) α enhanceosome activity and blocked TCR-Jα rearrangement. TLX1/TLX3 abrogation or enforced TCRαß expression leads to TCRα rearrangement and apoptosis. Importantly, the autoextinction of clones carrying TCRα-driven TLX1 expression supports TLX "addiction" in TLX-positive leukemias and provides further rationale for targeted therapy based on disruption of TLX1/TLX3.
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Regulación Neoplásica de la Expresión Génica , Genes Codificadores de la Cadena alfa de los Receptores de Linfocito T , Proteínas de Homeodominio/fisiología , Leucemia-Linfoma Linfoblástico de Células T Precursoras/genética , Proteína Proto-Oncogénica c-ets-1/metabolismo , Proteínas Proto-Oncogénicas/fisiología , Apoptosis , Sitios de Unión , Reordenamiento Génico , Células HeLa , Proteínas de Homeodominio/genética , Proteínas de Homeodominio/metabolismo , Humanos , Leucemia-Linfoma Linfoblástico de Células T Precursoras/patología , Estructura Terciaria de Proteína , Proteína Proto-Oncogénica c-ets-1/fisiología , Proteínas Proto-Oncogénicas/genética , Proteínas Proto-Oncogénicas/metabolismoRESUMEN
TLX1 is a homeodomain transcription factor generally associated with a favorable outcome in T-cell acute lymphoblastic leukemia (T-ALL). However, the molecular mechanisms of TLX1 deregulation remain unclear and various transcript levels in the absence of 10q24 abnormalities have been reported. A reproducible and accurate delineation of TLX1(+) T-ALL will be necessary for proper therapeutic stratification. We have studied 264 unselected T-ALLs (171 adults and 93 children) and show that T-ALLs expressing high levels of TLX1 (n = 35, 13%), defined as a real-time quantitative polymerase chain reaction (RQ-PCR) level of TLX1 greater than 1.00 ABL, form a homogeneous oncogenic group, based on their uniform stage of maturation arrest and oncogenetic and transcriptional profiles. Furthermore, TLX1-high T-ALLs harbor molecular TLX1 locus abnormalities in the majority (31/33), a proportion largely underestimated by standard karyotypic screening. T-ALLs expressing TLX1 at lower levels (n = 57, 22%) do not share these characteristics. Prognostic analysis within the adult LALA94 and GRAALL03 prospective protocols demonstrate a better event-free survival (P = .035) and a marked trend for longer overall survival (P = .059) for TLX1-high T-ALLs, while the expression of lower levels of TLX1 does not impact on prognosis. We propose that TLX1(+) T-ALLs be defined as cases expressing TLX1/ABL ratios greater than 1 and/or demonstrating TLX1 rearrangement. Therapeutic modification should be considered for those patients.
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Regulación Neoplásica de la Expresión Génica , Proteínas de Homeodominio/genética , Leucemia-Linfoma de Células T del Adulto/genética , Leucemia-Linfoma de Células T del Adulto/patología , Proteínas Proto-Oncogénicas/genética , Adolescente , Adulto , Alelos , Protocolos de Quimioterapia Combinada Antineoplásica , Cromosomas Humanos Par 10/genética , Femenino , Genotipo , Humanos , Inmunogenética , Cariotipificación , Leucemia-Linfoma de Células T del Adulto/tratamiento farmacológico , Masculino , Persona de Mediana Edad , Análisis de Secuencia por Matrices de Oligonucleótidos , Pronóstico , ARN Mensajero/genética , Tasa de Supervivencia , Transcripción Genética/genéticaRESUMEN
Activation of tyrosine kinase genes is a frequent event in human hematologic malignancies. Because gene activation could be associated with gene dysregulation, we attempted to screen for activating gene mutation based on high-level gene expression. We focused our study on the Janus kinase 2 (JAK2) gene in 90 cases of acute leukemia. This strategy led to the identification of a novel JAK2-acquired mutation in a patient with Down syndrome (DS) with B-cell precursor acute lymphoblastic leukemia (BCP-ALL). This mutation involves a 5-amino acid deletion within the JH2 pseudokinase domain (JAK2DeltaIREED). Expression of JAK2DeltaIREED in Ba/F3 cells induced constitutive activation of the JAK-STAT pathway and growth factor-independent cell proliferation. These results highlight the JAK2 pseudokinase domain as an oncogenic hot spot and indicate that activation of the JAK-STAT pathway may contribute to lymphoid malignancies and hematologic disorders observed in children with DS.
Asunto(s)
Linfocitos B/citología , Diferenciación Celular , Síndrome de Down/complicaciones , Janus Quinasa 2/genética , Janus Quinasa 2/metabolismo , Leucemia-Linfoma Linfoblástico de Células Precursoras/complicaciones , Leucemia-Linfoma Linfoblástico de Células Precursoras/enzimología , Secuencia de Aminoácidos , Animales , Linfocitos B/enzimología , Secuencia de Bases , Línea Celular Tumoral , Preescolar , Secuencia Conservada , Síndrome de Down/genética , Activación Enzimática/genética , Humanos , Janus Quinasa 2/química , Ratones , Datos de Secuencia Molecular , Mutación/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras/patología , Alineación de SecuenciaRESUMEN
The t(11;14)(p13;q11) is presumed to arise from an erroneous T-cell receptor delta TCRD V(D)J recombination and to result in LMO2 activation. However, the mechanisms underlying this translocation and the resulting LMO2 activation are poorly defined. We performed combined in vivo, ex vivo, and in silico analyses on 9 new t(11;14)(p13;q11)-positive T-cell acute lymphoblastic leukemia (T-ALL) as well as normal thymocytes. Our data support the involvement of 2 distinct t(11;14)(p13;q11) V(D)J-related translocation mechanisms. We provide compelling evidence that removal of a negative regulatory element from the LMO2 locus, rather than juxtaposition to the TCRD enhancer, is the main determinant for LMO2 activation in the majority of t(11;14)(p13;q11) translocations. Furthermore, the position of the LMO2 breakpoints in T-ALL in the light of the occurrence of TCRD-LMO2 translocations in normal thymocytes points to a critical role for the exact breakpoint location in determining LMO2 activation levels and the consequent pressure for T-ALL development.