RESUMEN
Cytogenetic analysis is mandatory at initial assessment of B-cell acute lymphoblastic leukemia (B-ALL) due to its diagnostic and prognostic value. Results from chromosome banding analysis and complementary FISH are taken into account in therapeutic protocols and further completed by other techniques (RT-PCR, SNP-array, MLPA, NGS, OGM). Indeed, new genomic entities have been identified by NGS, mostly RNA sequencing, such as Ph-like ALL that can benefit from targeted therapy. Here, we have attempted to establish cytogenetic guidelines by reviewing the most recent published data including the novel 5th World Health Organization and International Consensus Classifications. We also focused on newly described cytogenomic entities and indicate alternative diagnostic tools such as NGS technology, as its importance is vastly increasing in the diagnostic setting.
Asunto(s)
Hematología , Leucemia-Linfoma Linfoblástico de Células Precursoras , Humanos , Análisis Citogenético/métodos , Pronóstico , Sociedades MédicasRESUMEN
Molecular analysis is the hallmark of T-cell acute lymphoblastic leukemia (T-ALL) categorization. Several T-ALL sub-groups are well recognized based on the aberrant expression of specific transcription factors. This recently resulted in the implementation of eight provisional T-ALL entities into the novel 2022 International Consensus Classification, albeit not into the updated World Health Organization classification system. Despite this extensive molecular characterization, cytogenetic analysis remains the backbone of T-ALL diagnosis in many countries as chromosome banding analysis and fluorescence in situ hybridization are relatively inexpensive techniques to obtain results of diagnostic, prognostic and therapeutic interest. Here, we provide an overview of recurrent chromosomal abnormalities detectable in T-ALL patients and propose guidelines regarding their detection. By referring in parallel to the more general molecular classification approach, we hope to offer a diagnostic framework useful in a broad clinical genetic setting.
Asunto(s)
Hematología , Leucemia-Linfoma Linfoblástico de Células T Precursoras , Humanos , Hibridación Fluorescente in Situ , Leucemia-Linfoma Linfoblástico de Células T Precursoras/diagnóstico , Leucemia-Linfoma Linfoblástico de Células T Precursoras/genética , Leucemia-Linfoma Linfoblástico de Células T Precursoras/terapia , Análisis Citogenético/métodos , Linfocitos TAsunto(s)
Síndrome Hipereosinofílico , Linfoma , Trastornos Mieloproliferativos , Humanos , Receptor beta de Factor de Crecimiento Derivado de Plaquetas , Síndrome Hipereosinofílico/diagnóstico , Síndrome Hipereosinofílico/genética , Proteínas de Fusión Oncogénica/genética , Antígenos de Neoplasias , Proteínas de Unión al ARNRESUMEN
Acute lymphoblastic leukemia (ALL) is characterized by the presence of chromosomal changes, including numerical changes, translocations, and deletions, which are often associated with additional single-nucleotide mutations. In this study, we used single cell-targeted DNA sequencing to evaluate the clonal heterogeneity of B-ALL at diagnosis and during chemotherapy treatment. We designed a custom DNA amplicon library targeting mutational hotspot regions (in 110 genes) present in ALL, and we measured the presence of mutations and small insertions/deletions (indels) in bone marrow or blood samples from 12 B-ALL patients, with a median of 7973 cells per sample. Nine of the 12 cases showed at least 1 subclonal mutation, of which cases with PAX5 alterations or high hyperdiploidy (with intermediate to good prognosis) showed a high number of subclones (1 to 7) at diagnosis, defined by a variety of mutations in the JAK/STAT, RAS, or FLT3 signaling pathways. Cases with RAS pathway mutations had multiple mutations in FLT3, NRAS, KRAS, or BRAF in various clones. For those cases where we detected multiple mutational clones at diagnosis, we also studied blood samples during the first weeks of chemotherapy treatment. The leukemia clones disappeared during treatment with various kinetics, and few cells with mutations were easily detectable, even at low frequency (<0.1%). Our data illustrate that about half of the B-ALL cases show >2 subclones at diagnosis and that even very rare mutant cells can be detected at diagnosis or during treatment by single cell-targeted DNA sequencing.
RESUMEN
Acute lymphoblastic leukemia (ALL) is a malignancy that can be subdivided into distinct entities based on clinical, immunophenotypic and genomic features, including mutations, structural variants (SVs), and copy number alterations (CNA). Chromosome banding analysis (CBA) and Fluorescent In-Situ Hybridization (FISH) together with Multiple Ligation-dependent Probe Amplification (MLPA), array and PCR-based methods form the backbone of routine diagnostics. This approach is labor-intensive, time-consuming and costly. New molecular technologies now exist that can detect SVs and CNAs in one test. Here we apply one such technology, optical genome mapping (OGM), to the diagnostic work-up of 41 ALL cases. Compared to our standard testing pathway, OGM identified all recurrent CNAs and SVs as well as additional recurrent SVs and the resulting fusion genes. Based on the genomic profile obtained by OGM, 32 patients could be assigned to one of the major cytogenetic risk groups compared to 23 with the standard approach. The latter identified 24/34 recurrent chromosomal abnormalities, while OGM identified 33/34, misinterpreting only 1 case with low hypodiploidy. The results of MLPA were concordant in 100% of cases. Overall, there was excellent concordance between the results. OGM increased the detection rate and cytogenetic resolution, and abrogated the need for cascade testing, resulting in reduced turnaround times. OGM also provided opportunities for better patient stratification and accurate treatment options. However, for comprehensive cytogenomic testing, OGM still needs to be complemented with CBA or SNP-array to detect ploidy changes and with BCR::ABL1 FISH to assign patients as soon as possible to targeted therapy.
Asunto(s)
Aberraciones Cromosómicas , Leucemia-Linfoma Linfoblástico de Células Precursoras , Mapeo Cromosómico/métodos , Variaciones en el Número de Copia de ADN , Humanos , Leucemia-Linfoma Linfoblástico de Células Precursoras/diagnóstico , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , Flujo de TrabajoRESUMEN
Spi-1 Proto-Oncogene (SPI1) fusion genes are recurrently found in T-cell acute lymphoblastic leukemia (T-ALL) cases but are insufficient to drive leukemogenesis. Here we show that SPI1 fusions in combination with activating NRAS mutations drive an immature T-ALL in vivo using a conditional bone marrow transplant mouse model. Addition of the oncogenic fusion to the NRAS mutation also results in a higher leukemic stem cell frequency. Mechanistically, genetic deletion of the ß-catenin binding domain within Transcription factor 7 (TCF7)-SPI1 or use of a TCF/ß-catenin interaction antagonist abolishes the oncogenic activity of the fusion. Targeting the TCF7-SPI1 fusion in vivo with a doxycycline-inducible knockdown results in increased differentiation. Moreover, both pharmacological and genetic inhibition lead to down-regulation of SPI1 targets. Together, our results reveal an example where TCF7-SPI1 leukemia is vulnerable to pharmacological targeting of the TCF/ß-catenin interaction.
Asunto(s)
GTP Fosfohidrolasas/metabolismo , Proteínas de la Membrana/metabolismo , Leucemia-Linfoma Linfoblástico de Células T Precursoras/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Factor 1 de Transcripción de Linfocitos T/metabolismo , Transactivadores/metabolismo , beta Catenina/metabolismo , Animales , Trasplante de Médula Ósea , Carcinogénesis/genética , Modelos Animales de Enfermedad , Femenino , GTP Fosfohidrolasas/genética , Células HEK293 , Humanos , Proteínas de la Membrana/genética , Ratones , Ratones Endogámicos C57BL , Mutación , Proteínas de Fusión Oncogénica/genética , Proteínas de Fusión Oncogénica/metabolismo , Oncogenes , Leucemia-Linfoma Linfoblástico de Células T Precursoras/genética , Proto-Oncogenes Mas , Proteínas Proto-Oncogénicas/genética , Factor 1 de Transcripción de Linfocitos T/genética , Linfocitos T/metabolismo , Transactivadores/genética , Transcriptoma , beta Catenina/genéticaRESUMEN
T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive leukemia that is most frequent in children and is characterized by the presence of few chromosomal rearrangements and 10 to 20 somatic mutations in protein-coding regions at diagnosis. The majority of T-ALL cases harbor activating mutations in NOTCH1 together with mutations in genes implicated in kinase signaling, transcriptional regulation, or protein translation. To obtain more insight in the level of clonal heterogeneity at diagnosis and during treatment, we used single-cell targeted DNA sequencing with the Tapestri platform. We designed a custom ALL panel and obtained accurate single-nucleotide variant and small insertion-deletion mutation calling for 305 amplicons covering 110 genes in about 4400 cells per sample and time point. A total of 108 188 cells were analyzed for 25 samples of 8 T-ALL patients. We typically observed a major clone at diagnosis (>35% of the cells) accompanied by several minor clones of which some were less than 1% of the total number of cells. Four patients had >2 NOTCH1 mutations, some of which present in minor clones, indicating a strong pressure to acquire NOTCH1 mutations in developing T-ALL cells. By analyzing longitudinal samples, we detected the presence and clonal nature of residual leukemic cells and clones with a minor presence at diagnosis that evolved to clinically relevant major clones at later disease stages. Therefore, single-cell DNA amplicon sequencing is a sensitive assay to detect clonal architecture and evolution in T-ALL.
Asunto(s)
Evolución Clonal , ADN de Neoplasias/genética , Mutación , Leucemia-Linfoma Linfoblástico de Células T Precursoras/genética , Análisis de la Célula Individual/métodos , Células Sanguíneas/química , Células de la Médula Ósea/química , Niño , Humanos , Mutación INDEL , Masculino , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/fisiología , Neoplasia Residual/diagnóstico , Fosfohidrolasa PTEN/genética , Filogenia , Polimorfismo de Nucleótido Simple , Leucemia-Linfoma Linfoblástico de Células T Precursoras/patología , Receptor Notch1/genética , Receptor Notch1/fisiología , Recurrencia , Terapia Recuperativa , Sensibilidad y Especificidad , Análisis de Secuencia de ADNRESUMEN
PURPOSE: KPT-8602 (Eltanexor) is a second-generation exportin-1 (XPO1) inhibitor with potent activity against acute lymphoblastic leukemia (ALL) in preclinical models and with minimal effects on normal cells. In this study, we evaluated whether KPT-8602 would synergize with dexamethasone, vincristine, or doxorubicin, three drugs currently used for the treatment of ALL. EXPERIMENTAL DESIGN: First, we searched for the most synergistic combination of KPT-8602 with dexamethasone, vincristine, or doxorubicin in vitro in both B-ALL and T-ALL cell lines using proliferation and apoptosis as a readout. Next, we validated this synergistic effect by treatment of clinically relevant B- and T-ALL patient-derived xenograft models in vivo. Finally, we performed RNA-sequencing (RNA-seq) and chromatin immunoprecipitation sequencing (ChIP-seq) to determine the mechanism of synergy. RESULTS: KPT-8602 showed strong synergism with dexamethasone on human B-ALL and T-ALL cell lines as well as in vivo in three patient-derived ALL xenografts. Compared with single-drug treatment, the drug combination caused increased apoptosis and led to histone depletion. Mechanistically, integration of ChIP-seq and RNA-seq data revealed that addition of KPT-8602 to dexamethasone enhanced the activity of the glucocorticoid receptor (NR3C1) and led to increased inhibition of E2F-mediated transcription. We observed strong inhibition of E2F target genes related to cell cycle, DNA replication, and transcriptional regulation. CONCLUSIONS: Our preclinical study demonstrates that KPT-8602 enhances the effects of dexamethasone to inhibit B-ALL and T-ALL cells via NR3C1- and E2F-mediated transcriptional complexes, allowing to achieve increased dexamethasone effects for patients.
Asunto(s)
Dexametasona/farmacología , Doxorrubicina/farmacología , Carioferinas/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras/tratamiento farmacológico , Receptores Citoplasmáticos y Nucleares/genética , Animales , Antineoplásicos/farmacología , Apoptosis/efectos de los fármacos , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Sinergismo Farmacológico , Humanos , Carioferinas/antagonistas & inhibidores , Ratones , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras/patología , Receptores Citoplasmáticos y Nucleares/antagonistas & inhibidores , Vincristina/farmacología , Ensayos Antitumor por Modelo de Xenoinjerto , Proteína Exportina 1RESUMEN
The NUP214-ABL1 fusion is a constitutively activated tyrosine kinase that is significantly associated with overexpression of the TLX1 and TLX3 transcription factors in T cell acute lymphoblastic leukemia (T-ALL). Here we show that NUP214-ABL1 cooperates with TLX1 in driving T-ALL development using a transgenic mouse model and human T-ALL cells. Using integrated ChIP-sequencing, ATAC-sequencing, and RNA-sequencing data, we demonstrate that TLX1 and STAT5, the downstream effector of NUP214-ABL1, co-bind poised enhancer regions, and cooperatively activate the expression of key proto-oncogenes such as MYC and BCL2. Inhibition of STAT5, downregulation of TLX1 or MYC, or interference with enhancer function through BET-inhibitor treatment leads to reduction of target gene expression and induction of leukemia cell death.
Asunto(s)
Elementos de Facilitación Genéticos , Proteínas de Homeodominio/fisiología , Proteínas de Complejo Poro Nuclear/genética , Leucemia-Linfoma Linfoblástico de Células T Precursoras/genética , Proteínas Proto-Oncogénicas c-abl/genética , Proteínas Proto-Oncogénicas/fisiología , Factor de Transcripción STAT5/fisiología , Animales , Fusión Génica , Proteínas de Homeodominio/genética , Humanos , Ratones , Leucemia-Linfoma Linfoblástico de Células T Precursoras/patología , Proteínas Proto-Oncogénicas/genética , Proteínas Proto-Oncogénicas c-myc/fisiología , Factor de Transcripción STAT5/genéticaRESUMEN
Next-generation sequencing has provided a detailed overview of the various genomic lesions implicated in the pathogenesis of T-cell acute lymphoblastic leukemia (T-ALL). Typically, 10-20 protein-altering lesions are found in T-ALL cells at diagnosis. However, it is currently unclear in which order these mutations are acquired and in which progenitor cells this is initiated. To address these questions, we used targeted single-cell sequencing of total bone marrow cells and CD34+CD38- multipotent progenitor cells for four T-ALL cases. Hierarchical clustering detected a dominant leukemia cluster at diagnosis, accompanied by a few smaller clusters harboring only a fraction of the mutations. We developed a graph-based algorithm to determine the order of mutation acquisition. Two of the four patients had an early event in a known oncogene (MED12, STAT5B) among various pre-leukemic events. Intermediate events included loss of 9p21 (CDKN2A/B) and acquisition of fusion genes, while NOTCH1 mutations were typically late events. Analysis of CD34+CD38- cells and myeloid progenitors revealed that in half of the cases somatic mutations were detectable in multipotent progenitor cells. We demonstrate that targeted single-cell sequencing can elucidate the order of mutation acquisition in T-ALL and that T-ALL development can start in a multipotent progenitor cell.
Asunto(s)
Mutación , Leucemia-Linfoma Linfoblástico de Células T Precursoras/genética , ADP-Ribosil Ciclasa 1/análisis , Antígenos CD34/análisis , Perfilación de la Expresión Génica , Humanos , Células Madre Multipotentes/metabolismo , Receptor Notch1/genética , Secuenciación Completa del GenomaRESUMEN
Purpose: Human exportin-1 (XPO1) is the key nuclear-cytoplasmic transport protein that exports different cargo proteins out of the nucleus. Inducing nuclear accumulation of these proteins by inhibiting XPO1 causes cancer cell death. First clinical validation of pharmacological inhibition of XPO1 was obtained with the Selective Inhibitor of Nuclear Export (SINE) compound selinexor (KPT-330) demonstrating activity in phase-II/IIb clinical trials when dosed 1 to 3 times weekly. The second-generation SINE compound KPT-8602 shows improved tolerability and can be dosed daily. Here, we investigate and validate the drug-target interaction of KPT-8602 and explore its activity against acute lymphoblastic leukemia (ALL).Experimental Design: We examined the effect of KPT-8602 on XPO1 function and XPO1-cargo as well as on a panel of leukemia cell lines. Mutant XPO1 leukemia cells were designed to validate KPT-8602's drug-target interaction. In vivo, anti-ALL activity was measured in a mouse ALL model and patient-derived ALL xenograft models.Results: KPT-8602 induced caspase-dependent apoptosis in a panel of leukemic cell lines in vitro Using CRISPR/Cas9 genome editing, we demonstrated the specificity of KPT-8602 for cysteine 528 in the cargo-binding groove of XPO1 and validated the drug target interaction. In vivo, KPT-8602 showed potent anti-leukemia activity in a mouse ALL model as well as in patient-derived T- and B-ALL xenograft models without affecting normal hematopoiesis.Conclusions: KPT-8602 is highly specific for XPO1 inhibition and demonstrates potent anti-leukemic activity supporting clinical application of the second-generation SINE compound for the treatment of ALL. Clin Cancer Res; 23(10); 2528-41. ©2016 AACR.
Asunto(s)
Antineoplásicos/administración & dosificación , Carioferinas/antagonistas & inhibidores , Leucemia-Linfoma Linfoblástico de Células Precursoras/tratamiento farmacológico , Receptores Citoplasmáticos y Nucleares/antagonistas & inhibidores , Tiazoles/administración & dosificación , Transporte Activo de Núcleo Celular/efectos de los fármacos , Animales , Antineoplásicos/química , Apoptosis/efectos de los fármacos , Sistemas CRISPR-Cas , Línea Celular Tumoral , Edición Génica , Humanos , Carioferinas/genética , Ratones , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras/patología , Receptores Citoplasmáticos y Nucleares/genética , Ensayos Antitumor por Modelo de Xenoinjerto , Proteína Exportina 1RESUMEN
T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive childhood leukemia that is caused by the accumulation of multiple genomic lesions resulting in transcriptional deregulation and increased cell proliferation and survival. Through analysis of gene expression data, we provide evidence that the hedgehog pathway is activated in 20% of T-ALL samples. Hedgehog pathway activation is associated with ectopic expression of the hedgehog ligands Sonic hedgehog (SHH) or Indian hedgehog (IHH), and with upregulation of the transcription factor GLI1 Ectopic expression of SHH or IHH in mouse T cells in vivo caused hedgehog pathway activation in both lymphoid and epithelial cells in the thymus and resulted in increased expression of important T-cell stimulatory ligands (Dll4, Il7, and Vegf) by thymic epithelial cells. In T-ALL cell lines, pharmacological inhibition or short interfering RNA-mediated knockdown of SMO or GLI1 led to decreased cell proliferation. Moreover, primary T-ALL cases with high GLI1 messenger RNA levels, but not those with low or undetectable GLI1 expression, were sensitive to hedgehog pathway inhibition by GANT61 or GDC-0449 (vismodegib) using ex vivo cultures and in vivo xenograft models. We identify the hedgehog pathway as a novel therapeutic target in T-ALL and demonstrate that hedgehog inhibitors approved by the US Food and Drug Administration could be used for the treatment of this rare leukemia.