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ABSTRACT: Acute lymphoblastic leukemia (ALL) with fusions of ABL-class tyrosine kinase genes other than BCR::ABL1 occurs in â¼3% of children with ALL. The tyrosine kinase genes involved in this BCR::ABL1-like (Ph-like) subtype include ABL1, PDGFRB, ABL2, and CSF1R, each of which has up to 10 described partner genes. ABL-class ALL resembles BCR::ABL1-positive ALL with a similar gene expression profile, poor response to chemotherapy, and sensitivity to tyrosine kinase inhibitors (TKIs). There is a lack of comprehensive data regarding TKI sensitivity in the heterogeneous group of ABL-class ALL. We observed variability in TKI sensitivity within and among each ABL-class tyrosine kinase gene subgroup. We showed that ALL samples with fusions for any of the 4 tyrosine kinase genes were relatively sensitive to imatinib. In contrast, the PDGFRB-fused ALL samples were less sensitive to dasatinib and bosutinib. Variation in ex vivo TKI response within the subset of samples with the same ABL-class tyrosine kinase gene was not associated with the ALL immunophenotype, 5' fusion partner, presence or absence of Src-homology-2/3 domains, or deletions of IKZF1, PAX5, or CDKN2A/B. In conclusion, the tyrosine kinase gene involved in ABL-class ALL is the main determinant of TKI sensitivity and relevant for specific TKI selection.
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Leucemia-Linfoma Linfoblástico de Células Precursoras , Inhibidores de Proteínas Quinasas , Proteínas Proto-Oncogénicas c-abl , Dominios Homologos src , Humanos , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras/tratamiento farmacológico , Leucemia-Linfoma Linfoblástico de Células Precursoras/patología , Inhibidores de Proteínas Quinasas/uso terapéutico , Inhibidores de Proteínas Quinasas/farmacología , Niño , Proteínas Proto-Oncogénicas c-abl/genética , Proteínas Proto-Oncogénicas c-abl/metabolismo , Adolescente , Preescolar , Femenino , Masculino , Mesilato de Imatinib/uso terapéutico , Mesilato de Imatinib/farmacología , Receptor beta de Factor de Crecimiento Derivado de Plaquetas/genética , Receptor beta de Factor de Crecimiento Derivado de Plaquetas/metabolismo , Dasatinib/uso terapéutico , Dasatinib/farmacología , Proteínas de Fusión Oncogénica/genéticaRESUMEN
ABSTRACT: Pediatric B-cell precursor (BCP) lymphoblastic malignancies are neoplasms with manifestation either in the bone marrow or blood (BCP acute lymphoblastic leukemia [BCP-ALL]) or are less common in extramedullary tissue (BCP lymphoblastic lymphoma [BCP-LBL]). Although both presentations are similar in morphology and immunophenotype, molecular studies have been virtually restricted to BCP-ALL so far. The lack of molecular studies on BCP-LBL is due to its rarity and restriction on small, mostly formalin-fixed paraffin-embedded (FFPE) tissues. Here, to our knowledge, we present the first comprehensive mutational and transcriptional analysis of what we consider the largest BCP-LBL cohort described to date (n = 97). Whole-exome sequencing indicated a mutational spectrum of BCP-LBL, strikingly similar to that found in BCP-ALL. However, epigenetic modifiers were more frequently mutated in BCP-LBL, whereas BCP-ALL was more frequently affected by mutation in genes involved in B-cell development. Integrating copy number alterations, somatic mutations, and gene expression by RNA sequencing revealed that virtually all molecular subtypes originally defined in BCP-ALL are present in BCP-LBL, with only 7% of lymphomas that were not assigned to a subtype. Similar to BCP-ALL, the most frequent subtypes of BCP-LBL were high hyperdiploidy and ETV6::RUNX1. Tyrosine kinase/cytokine receptor rearrangements were detected in 7% of BCP-LBL. These results indicate that genetic subtypes can be identified in BCP-LBL using next-generation sequencing, even in FFPE tissue, and may be relevant to guide treatment.
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Mutación , Leucemia-Linfoma Linfoblástico de Células Precursoras B , Humanos , Niño , Leucemia-Linfoma Linfoblástico de Células Precursoras B/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras B/patología , Masculino , Preescolar , Femenino , Adolescente , Lactante , Secuenciación del Exoma , Transcripción GenéticaRESUMEN
ABSTRACT: Acute lymphoblastic leukemia (ALL) arises from the uncontrolled proliferation of B-cell precursors (BCP-ALL) or T cells (T-ALL). Current treatment protocols obtain high cure rates in children but are based on toxic polychemotherapy. Novel therapies are urgently needed, especially in relapsed/refractory (R/R) disease, high-risk (HR) leukemias and T-ALL, in which immunotherapy approaches remain scarce. Although the interleukin-7 receptor (IL-7R) plays a pivotal role in ALL development, no IL-7R-targeting immunotherapy has yet reached clinical application in ALL. The IL-7Rα chain (CD127)-targeting IgG4 antibody lusvertikimab (LUSV; formerly OSE-127) is a full antagonist of the IL-7R pathway, showing a good safety profile in healthy volunteers. Here, we show that â¼85% of ALL cases express surface CD127. We demonstrate significant in vivo efficacy of LUSV immunotherapy in a heterogeneous cohort of BCP- and T-ALL patient-derived xenografts (PDX) in minimal residual disease (MRD) and overt leukemia models, including R/R and HR leukemias. Importantly, LUSV was particularly effective when combined with polychemotherapy in a phase 2-like PDX study with CD127high samples leading to MRD-negativity in >50% of mice treated with combination therapy. Mechanistically, LUSV targeted ALL cells via a dual mode of action comprising direct IL-7R antagonistic activity and induction of macrophage-mediated antibody-dependent cellular phagocytosis (ADCP). LUSV-mediated in vitro ADCP levels significantly correlated with CD127 expression levels and the reduction of leukemia burden upon treatment of PDX animals in vivo. Altogether, through its dual mode of action and good safety profile, LUSV may represent a novel immunotherapy option for any CD127+ ALL, particularly in combination with standard-of-care polychemotherapy.
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Ensayos Antitumor por Modelo de Xenoinjerto , Animales , Humanos , Ratones , Receptores de Interleucina-7/antagonistas & inhibidores , Ratones SCID , Fagocitosis/efectos de los fármacos , Subunidad alfa del Receptor de Interleucina-7 , Leucemia-Linfoma Linfoblástico de Células Precursoras/tratamiento farmacológico , Leucemia-Linfoma Linfoblástico de Células Precursoras/patología , Leucemia-Linfoma Linfoblástico de Células Precursoras/inmunología , Femenino , Ratones Endogámicos NOD , Anticuerpos Monoclonales Humanizados/uso terapéutico , Anticuerpos Monoclonales Humanizados/farmacología , Línea Celular Tumoral , Antineoplásicos Inmunológicos/farmacología , Antineoplásicos Inmunológicos/uso terapéuticoRESUMEN
A common finding in pediatric B-cell precursor acute lymphoblastic leukemia (BCPALL) is that chromosome 21 is never lost and an extra chromosome 21 is often gained. This implies an important role for chromosome 21 in the pathobiology of BCPALL, emphasized by the increased risk of BCPALL in children with Down syndrome. However, model systems of chromosome 21 gain are lacking. We therefore developed a BCPALL cell line (Nalm-6, DUX4-rearranged) with an additional chromosome 21 by means of microcell-mediated chromosome transfer. FISH, PCR, multiplex ligation-dependent probe amplification, and whole exome sequencing showed that an additional chromosome 21 was successfully transferred to the recipient cells. Transcription of some but not all genes on chromosome 21 was increased, indicating tight transcriptional regulation. Nalm-6 cells with an additional chromosome 21 proliferated slightly slower compared with parental Nalm-6 and sensitivity to induction chemotherapeutics was mildly increased. The extra copy of chromosome 21 did not confer sensitivity to targeted signaling inhibitors. In conclusion, a BCPALL cell line with an additional human chromosome 21 was developed, validated, and subjected to functional studies, which showed a minor but potentially relevant effect in vitro. This cell line offers the possibility to study further the role of chromosome 21 in ALL.
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Linfoma de Burkitt , Leucemia-Linfoma Linfoblástico de Células Precursoras B , Humanos , Niño , Cromosomas Humanos Par 21/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras B/genética , Reacción en Cadena de la PolimerasaRESUMEN
IKZF1-deletions occur in 10-15% of patients with B-cell precursor acute lymphoblastic leukemia (BCP-ALL) and predict a poor outcome. However, the impact of IKZF1-loss on sensitivity to drugs used in contemporary treatment protocols has remained underexplored. Here we show in experimental models and in patients that loss of IKZF1 promotes resistance to AraC, a key component of both upfront and relapsed treatment protocols. We attribute this resistance, in part, to diminished import and incorporation of cytarabine (AraC) due to reduced expression of the solute carrier hENT1. Moreover, we find elevated mRNA expression of Evi1, a known driver of therapy resistance in myeloid malignancies. Finally, a kinase directed CRISPR/Cas9-screen identified that inhibition of either mediator kinases CDK8/19 or casein kinase 2 can restore response to AraC. We conclude that this high-risk patient group could benefit from alternative antimetabolites, or targeted therapies that resensitize the cells to AraC.
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We previously identified an association of rapid engraftment of patient-derived leukemia cells transplanted into NOD/SCID mice with early relapse in B-cell precursor acute lymphoblastic leukemia (BCP-ALL). In a search for the cellular and molecular profiles associated with this phenotype, we investigated the expression of microRNAs (miRNAs) in different engraftment phenotypes and patient outcomes. We found high expression of miR-497 and miR-195 (hereafter miR-497/195) in patient-derived xenograft samples with slow engraftment derived from patients with favorable outcome. In contrast, epigenetic repression and low expression of these miRNAs was observed in rapidly engrafting samples associated with early relapse. Overexpression of miR-497/195 in patient-derived leukemia cells suppressed in vivo growth of leukemia and prolonged recipient survival. Conversely, inhibition of miR-497/195 led to increased leukemia cell growth. Key cell cycle regulators were downregulated upon miR-497/195 overexpression, and we identified cyclin-dependent kinase 4 (CDK4)- and cyclin-D3 (CCND3)-mediated control of G1/S transition as a principal mechanism for the suppression of BCP-ALL progression by miR-497/195. The critical role for miR-497/195-mediated cell cycle regulation was underscored by finding (in an additional independent series of patient samples) that high expression of miR-497/195 together with a full sequence for CDKN2A and CDKN2B (CDKN2A/B) was associated with excellent outcome, whereas deletion of CDKN2A/B together with low expression of miR-497/195 was associated with clearly inferior relapse-free survival. These findings point to the cooperative loss of cell cycle regulators as a new prognostic factor indicating possible therapeutic targets for pediatric BCP-ALL.
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Inhibidor p15 de las Quinasas Dependientes de la Ciclina/genética , Inhibidor p16 de la Quinasa Dependiente de Ciclina/genética , MicroARNs/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , Animales , Niño , Epigénesis Genética , Regulación Leucémica de la Expresión Génica , Humanos , Ratones Endogámicos NOD , Ratones SCID , Células Tumorales CultivadasRESUMEN
Risk stratification is essential for the delivery of optimal treatment in childhood acute lymphoblastic leukemia. However, current risk stratification algorithms dichotomize variables and apply risk factors independently, which may incorrectly assume identical associations across biologically heterogeneous subsets and reduce statistical power. Accordingly, we developed and validated a prognostic index (PIUKALL) that integrates multiple risk factors and uses continuous data. We created discovery (n = 2405) and validation (n = 2313) cohorts using data from 4 recent trials (UKALL2003, COALL-03, DCOG-ALL10, and NOPHO-ALL2008). Using the discovery cohort, multivariate Cox regression modeling defined a minimal model including white cell count at diagnosis, pretreatment cytogenetics, and end-of-induction minimal residual disease. Using this model, we defined PIUKALL as a continuous variable that assigns personalized risk scores. PIUKALL correlated with risk of relapse and was validated in an independent cohort. Using PIUKALL to risk stratify patients improved the concordance index for all end points compared with traditional algorithms. We used PIUKALL to define 4 clinically relevant risk groups that had differential relapse rates at 5 years and were similar between the 2 cohorts (discovery: low, 3% [95% confidence interval (CI), 2%-4%]; standard, 8% [95% CI, 6%-10%]; intermediate, 17% [95% CI, 14%-21%]; and high, 48% [95% CI, 36%-60%; validation: low, 4% [95% CI, 3%-6%]; standard, 9% [95% CI, 6%-12%]; intermediate, 17% [95% CI, 14%-21%]; and high, 35% [95% CI, 24%-48%]). Analysis of the area under the curve confirmed the PIUKALL groups were significantly better at predicting outcome than algorithms employed in each trial. PIUKALL provides an accurate method for predicting outcome and more flexible method for defining risk groups in future studies.
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Biomarcadores de Tumor/análisis , Recurrencia Local de Neoplasia/patología , Neoplasia Residual/patología , Evaluación de Resultado en la Atención de Salud/estadística & datos numéricos , Selección de Paciente , Leucemia-Linfoma Linfoblástico de Células Precursoras/patología , Adolescente , Niño , Preescolar , Terapia Combinada , Femenino , Estudios de Seguimiento , Humanos , Lactante , Masculino , Recurrencia Local de Neoplasia/terapia , Neoplasia Residual/terapia , Leucemia-Linfoma Linfoblástico de Células Precursoras/terapia , Pronóstico , Estudios Retrospectivos , Factores de Riesgo , Tasa de SupervivenciaRESUMEN
Translocation t(12;21), resulting in the ETV6-RUNX1 (or TEL-AML1) fusion protein, is present in 25% of pediatric patients with B-cell precursor acute lymphoblastic leukemia and is considered a first hit in leukemogenesis. A targeted therapy approach is not available for children with this subtype of leukemia. To identify the molecular mechanisms underlying ETV6-RUNX1-driven leukemia, we performed gene expression profiling of healthy hematopoietic progenitors in which we ectopically expressed ETV6-RUNX1. We reveal an ETV6-RUNX1-driven transcriptional network that induces proliferation, survival and cellular homeostasis. In addition, Vps34, an important regulator of autophagy, was found to be induced by ETV6-RUNX1 and up-regulated in ETV6-RUNX1-positive leukemic patient cells. We show that induction of Vps34 was transcriptionally regulated by ETV6-RUNX1 and correlated with high levels of autophagy. Knockdown of Vps34 in ETV6-RUNX1-positive cell lines severely reduced proliferation and survival. Inhibition of autophagy by hydroxychloroquine, a well-tolerated autophagy inhibitor, reduced cell viability in both ETV6-RUNX1-positive cell lines and primary acute lymphoblastic leukemia samples, and selectively sensitized primary ETV6-RUNX1-positive leukemia samples to L asparaginase. These findings reveal a causal relationship between ETV6-RUNX1 and autophagy, and provide pre-clinical evidence for the efficacy of autophagy inhibitors in ETV6-RUNX1-driven leukemia.
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Asparaginasa/farmacología , Muerte Celular Autofágica/efectos de los fármacos , Subunidad alfa 2 del Factor de Unión al Sitio Principal/metabolismo , Sistemas de Liberación de Medicamentos , Proteínas de Fusión Oncogénica/metabolismo , Leucemia-Linfoma Linfoblástico de Células Precursoras B/tratamiento farmacológico , Muerte Celular Autofágica/genética , Niño , Preescolar , Fosfatidilinositol 3-Quinasas Clase III/genética , Fosfatidilinositol 3-Quinasas Clase III/metabolismo , Subunidad alfa 2 del Factor de Unión al Sitio Principal/genética , Femenino , Humanos , Masculino , Proteínas de Fusión Oncogénica/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras B/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras B/metabolismo , Leucemia-Linfoma Linfoblástico de Células Precursoras B/patologíaRESUMEN
To prevent relapse, high risk paediatric acute lymphoblastic leukaemia (ALL) is treated very intensively. However, most patients who eventually relapse have standard or medium risk ALL with low minimal residual disease (MRD) levels. We analysed recurrent microdeletions and other clinical prognostic factors in a cohort of 475 uniformly treated non-high risk precursor B-cell ALL patients with the aim of better predicting relapse and refining risk stratification. Lower relapse-free survival at 7 years (RFS) was associated with IKZF1 intragenic deletions (P < 0·0001); P2RY8-CRLF2 gene fusion (P < 0·0004); Day 33 MRD>5 × 10-5 (P < 0·0001) and High National Cancer Institute (NCI) risk (P < 0·0001). We created a predictive model based on a risk score (RS) for deletions, MRD and NCI risk, extending from an RS of 0 (RS0) for patients with no unfavourable factors to RS2 + for patients with 2 or 3 high risk factors. RS0, RS1, and RS2 + groups had RFS of 93%, 78% and 49%, respectively, and overall survival (OS) of 99%, 91% and 71%. The RS provided greater discrimination than MRD-based risk stratification into standard (89% RFS, 96% OS) and medium risk groups (79% RFS, 91% OS). We conclude that this RS may enable better early therapeutic stratification and thus improve cure rates for childhood ALL.
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Deleción Cromosómica , Leucemia-Linfoma Linfoblástico de Células Precursoras B/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras B/mortalidad , Eliminación de Secuencia , Adolescente , Factores de Edad , Biomarcadores de Tumor , Niño , Preescolar , Femenino , Genotipo , Humanos , Lactante , Masculino , Neoplasia Residual/diagnóstico , Leucemia-Linfoma Linfoblástico de Células Precursoras B/diagnóstico , Pronóstico , Modelos de Riesgos Proporcionales , Recurrencia , Medición de Riesgo , Factores de RiesgoAsunto(s)
Proteínas de Unión al ADN/genética , Proteínas de Fusión Oncogénica/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras B/genética , Receptores de Factor Estimulante de Colonias de Granulocitos y Macrófagos/genética , Adolescente , Adulto , Niño , Preescolar , Femenino , Humanos , Masculino , Translocación Genética , Adulto JovenAsunto(s)
Leucemia-Linfoma Linfoblástico de Células Precursoras , Humanos , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , Factores de Transcripción , Línea Celular , Proteínas de Fusión Oncogénica/genética , Proteínas de Fusión Oncogénica/metabolismo , Factores de Transcripción MEF2/genética , Factores de Transcripción MEF2/metabolismoRESUMEN
Deletions and mutations affecting lymphoid transcription factor IKZF1 (IKAROS) are associated with an increased relapse risk and poor outcome in B-cell precursor acute lymphoblastic leukemia. However, additional genetic events may either enhance or negate the effects of IKZF1 deletions on prognosis. In a large discovery cohort of 533 childhood B-cell precursor acute lymphoblastic leukemia patients, we observed that single-copy losses of BTG1 were significantly enriched in IKZF1-deleted B-cell precursor acute lymphoblastic leukemia (P=0.007). While BTG1 deletions alone had no impact on prognosis, the combined presence of BTG1 and IKZF1 deletions was associated with a significantly lower 5-year event-free survival (P=0.0003) and a higher 5-year cumulative incidence of relapse (P=0.005), when compared with IKZF1-deleted cases without BTG1 aberrations. In contrast, other copy number losses commonly observed in B-cell precursor acute lymphoblastic leukemia, such as CDKN2A/B, PAX5, EBF1 or RB1, did not affect the outcome of IKZF1-deleted acute lymphoblastic leukemia patients. To establish whether the combined loss of IKZF1 and BTG1 function cooperate in leukemogenesis, Btg1-deficient mice were crossed onto an Ikzf1 heterozygous background. We observed that loss of Btg1 increased the tumor incidence of Ikzf1+/- mice in a dose-dependent manner. Moreover, murine B cells deficient for Btg1 and Ikzf1+/- displayed increased resistance to glucocorticoids, but not to other chemotherapeutic drugs. Together, our results identify BTG1 as a tumor suppressor in leukemia that, when deleted, strongly enhances the risk of relapse in IKZF1-deleted B-cell precursor acute lymphoblastic leukemia, and augments the glucocorticoid resistance phenotype mediated by the loss of IKZF1 function.
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Transformación Celular Neoplásica/genética , Epistasis Genética , Factor de Transcripción Ikaros/genética , Proteínas de Neoplasias/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras B/genética , Proteínas Supresoras de Tumor/genética , Adolescente , Animales , Biomarcadores de Tumor , Transformación Celular Neoplásica/metabolismo , Niño , Preescolar , Modelos Animales de Enfermedad , Resistencia a Antineoplásicos/genética , Femenino , Eliminación de Gen , Predisposición Genética a la Enfermedad , Humanos , Factor de Transcripción Ikaros/metabolismo , Masculino , Ratones , Ratones Noqueados , Proteínas de Neoplasias/metabolismo , Evaluación del Resultado de la Atención al Paciente , Leucemia-Linfoma Linfoblástico de Células Precursoras B/diagnóstico , Leucemia-Linfoma Linfoblástico de Células Precursoras B/mortalidad , Pronóstico , Recurrencia , Proteínas Supresoras de Tumor/metabolismoRESUMEN
BACKGROUND: It has been shown that a random-effects framework can be used to test the association between a gene's expression level and the number of DNA copies of a set of genes. This gene-set modelling framework was later applied to find associations between mRNA expression and microRNA expression, by defining the gene sets using target prediction information. METHODS AND RESULTS: Here, we extend the model introduced by Menezes et al. 2009 to consider the effect of not just copy number, but also of other molecular profiles such as methylation changes and loss-of-heterozigosity (LOH), on gene expression levels. We will consider again sets of measurements, to improve robustness of results and increase the power to find associations. Our approach can be used genome-wide to find associations and yields a test to help separate true associations from noise. We apply our method to colon and to breast cancer samples, for which genome-wide copy number, methylation and gene expression profiles are available. Our findings include interesting gene expression-regulating mechanisms, which may involve only one of copy number or methylation, or both for the same samples. We even are able to find effects due to different molecular mechanisms in different samples. CONCLUSIONS: Our method can equally well be applied to cases where other types of molecular (high-dimensional) data are collected, such as LOH, SNP genotype and microRNA expression data. Computationally efficient, it represents a flexible and powerful tool to study associations between high-dimensional datasets. The method is freely available via the SIM BioConductor package.
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Neoplasias de la Mama/genética , Neoplasias del Colon/genética , Biología Computacional/métodos , Regulación de la Expresión Génica , Redes Reguladoras de Genes , Transcriptoma , Simulación por Computador , Metilación de ADN , Femenino , Dosificación de Gen , Genotipo , Humanos , Pérdida de Heterocigocidad , Polimorfismo de Nucleótido SimpleRESUMEN
Persistent infection of basal keratinocytes with high-risk human papillomavirus (hrHPV) may cause cancer. Keratinocytes are equipped with different pattern recognition receptors (PRRs) but hrHPV has developed ways to dampen their signals resulting in minimal inflammation and evasion of host immunity for sustained periods of time. To understand the mechanisms underlying hrHPV's capacity to evade immunity, we studied PRR signaling in non, newly, and persistently hrHPV-infected keratinocytes. We found that active infection with hrHPV hampered the relay of signals downstream of the PRRs to the nucleus, thereby affecting the production of type-I interferon and pro-inflammatory cytokines and chemokines. This suppression was shown to depend on hrHPV-induced expression of the cellular protein ubiquitin carboxyl-terminal hydrolase L1 (UCHL1) in keratinocytes. UCHL1 accomplished this by inhibiting tumor necrosis factor receptor-associated factor 3 (TRAF3) K63 poly-ubiquitination which lead to lower levels of TRAF3 bound to TANK-binding kinase 1 and a reduced phosphorylation of interferon regulatory factor 3. Furthermore, UCHL1 mediated the degradation of the NF-kappa-B essential modulator with as result the suppression of p65 phosphorylation and canonical NF-κB signaling. We conclude that hrHPV exploits the cellular protein UCHL1 to evade host innate immunity by suppressing PRR-induced keratinocyte-mediated production of interferons, cytokines and chemokines, which normally results in the attraction and activation of an adaptive immune response. This identifies UCHL1 as a negative regulator of PRR-induced immune responses and consequently its virus-increased expression as a strategy for hrHPV to persist.
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Regulación Enzimológica de la Expresión Génica/inmunología , Papillomavirus Humano 16/inmunología , Inmunidad Innata , Queratinocitos/inmunología , Infecciones por Papillomavirus/inmunología , Ubiquitina Tiolesterasa/inmunología , Regulación hacia Arriba/inmunología , Células 3T3 , Animales , Células Cultivadas , Citocinas/inmunología , Citocinas/metabolismo , Femenino , Regulación Viral de la Expresión Génica/inmunología , Papillomavirus Humano 16/metabolismo , Humanos , Queratinocitos/enzimología , Queratinocitos/patología , Queratinocitos/virología , Ratones , Infecciones por Papillomavirus/enzimología , Infecciones por Papillomavirus/patología , Fosforilación/inmunología , Proteínas Serina-Treonina Quinasas/inmunología , Proteínas Serina-Treonina Quinasas/metabolismo , Transducción de Señal/inmunología , Factor 3 Asociado a Receptor de TNF/inmunología , Factor 3 Asociado a Receptor de TNF/metabolismo , Factor de Transcripción ReIA/inmunología , Factor de Transcripción ReIA/metabolismo , Ubiquitina Tiolesterasa/biosíntesis , Ubiquitinación/inmunología , Neoplasias del Cuello Uterino/inmunología , Neoplasias del Cuello Uterino/metabolismo , Neoplasias del Cuello Uterino/patología , Neoplasias del Cuello Uterino/virologíaRESUMEN
Current microRNA target predictions are based on sequence information and empirically derived rules but do not make use of the expression of microRNAs and their targets. This study aimed to improve microRNA target predictions in a given biological context, using in silico predictions, microRNA and mRNA expression. We used target prediction tools to produce lists of predicted targets and used a gene set test designed to detect consistent effects of microRNAs on the joint expression of multiple targets. In a single test, association between microRNA expression and target gene set expression as well as the contribution of the individual target genes on the association are determined. The strongest negatively associated mRNAs as measured by the test were prioritized. We applied our integration method to a well-defined muscle differentiation model. Validation of our predictions in C2C12 cells confirmed predicted targets of known as well as novel muscle-related microRNAs. We further studied associations between microRNA-mRNA pairs in human prostate cancer, finding some pairs that have been recently experimentally validated by others. Using the same study, we showed the advantages of the global test over Pearson correlation and lasso. We conclude that our integrated approach successfully identifies regulated microRNAs and their targets.
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Regulación Neoplásica de la Expresión Génica , MicroARNs/análisis , Mioblastos Esqueléticos/metabolismo , ARN Mensajero/análisis , Programas Informáticos , Regiones no Traducidas 3' , Algoritmos , Animales , Diferenciación Celular , Humanos , Masculino , Ratones , MicroARNs/genética , Mioblastos Esqueléticos/citología , Neoplasias de la Próstata/metabolismo , Neoplasias de la Próstata/patología , ARN Mensajero/genética , TranscriptomaRESUMEN
BACKGROUND: A number of statistical models has been proposed for studying the association between gene expression and copy number data in integrated analysis. The next step is to compare association patterns between different groups of samples. RESULTS: We propose a method, named dSIM, to find differences in association between copy number and gene expression, when comparing two groups of samples. Firstly, we use ridge regression to correct for the baseline associations between copy number and gene expression. Secondly, the global test is applied to the corrected data in order to find differences in association patterns between two groups of samples. We show that dSIM detects differences even in small genomic regions in a simulation study. We also apply dSIM to two publicly available breast cancer datasets and identify chromosome arms where copy number led gene expression regulation differs between positive and negative estrogen receptor samples. In spite of differing genomic coverage, some selected arms are identified in both datasets. CONCLUSION: We developed a flexible and robust method for studying association differences between two groups of samples while integrating genomic data from different platforms. dSIM can be used with most types of microarray/sequencing data, including methylation and microRNA expression. The method is implemented in R and will be made part of the BioConductor package SIM.
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Biología Computacional/métodos , Perfilación de la Expresión Génica , Neoplasias de la Mama/genética , Neoplasias de la Mama/metabolismo , Femenino , Dosificación de Gen/genética , Humanos , Receptores de Estrógenos/metabolismoAsunto(s)
Regulación Leucémica de la Expresión Génica , Proteínas de Neoplasias , Proteínas Nucleares , Proteínas de Fusión Oncogénica , Leucemia-Linfoma Linfoblástico de Células Precursoras B , Leucemia-Linfoma Linfoblástico de Células Precursoras , Niño , Bandeo Cromosómico , Cromosomas Humanos , Femenino , Humanos , Masculino , Proteínas de Neoplasias/biosíntesis , Proteínas de Neoplasias/genética , Proteínas Nucleares/biosíntesis , Proteínas Nucleares/genética , Proteínas de Fusión Oncogénica/biosíntesis , Proteínas de Fusión Oncogénica/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras B/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras B/metabolismo , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras/metabolismo , Leucemia-Linfoma Linfoblástico de Células Precursoras/patologíaRESUMEN
BACKGROUND: Various CACNA1A missense mutations cause familial hemiplegic migraine type 1 (FHM1), a rare monogenic subtype of migraine with aura. FHM1 mutation R192Q is associated with pure hemiplegic migraine, whereas the S218L mutation causes hemiplegic migraine, cerebellar ataxia, seizures, and mild head trauma-induced brain edema. Transgenic knock-in (KI) migraine mouse models were generated that carried either the FHM1 R192Q or the S218L mutation and were shown to exhibit increased CaV2.1 channel activity. Here we investigated their cerebellar and caudal cortical transcriptome. METHODS: Caudal cortical and cerebellar RNA expression profiles from mutant and wild-type mice were studied using microarrays. Respective brain regions were selected based on their relevance to migraine aura and ataxia. Relevant expression changes were further investigated at RNA and protein level by quantitative polymerase chain reaction (qPCR) and/or immunohistochemistry, respectively. RESULTS: Expression differences in the cerebellum were most pronounced in S218L mice. Particularly, tyrosine hydroxylase, a marker of delayed cerebellar maturation, appeared strongly upregulated in S218L cerebella. In contrast, only minimal expression differences were observed in the caudal cortex of either mutant mice strain. CONCLUSION: Despite pronounced consequences of migraine gene mutations at the neurobiological level, changes in cortical RNA expression in FHM1 migraine mice compared to wild-type are modest. In contrast, pronounced RNA expression changes are seen in the cerebellum of S218L mice and may explain their cerebellar ataxia phenotype.
Asunto(s)
Encéfalo/fisiopatología , Canales de Calcio Tipo N/genética , Ataxia Cerebelosa/genética , Cerebelo/fisiopatología , Corteza Cerebral/fisiopatología , Trastornos Migrañosos/genética , Proteínas del Tejido Nervioso/genética , ARN/metabolismo , Animales , Ataxia Cerebelosa/metabolismo , Femenino , Perfilación de la Expresión Génica , Regulación de la Expresión Génica/genética , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Transgénicos , Trastornos Migrañosos/metabolismo , Mutación , ARN/genética , Distribución Tisular , Transcriptoma/genéticaRESUMEN
In the design of microarray or next-generation sequencing experiments it is crucial to choose the appropriate number of biological replicates. As often the number of differentially expressed genes and their effect sizes are small and too few replicates will lead to insufficient power to detect these. On the other hand, too many replicates unnecessary leads to high experimental costs. Power and sample size analysis can guide experimentalist in choosing the appropriate number of biological replicates. Several methods for power and sample size analysis have recently been proposed for microarray data. However, most of these are restricted to two group comparisons and require user-defined effect sizes. Here we propose a pilot-data based method for power and sample size analysis which can handle more general experimental designs and uses pilot-data to obtain estimates of the effect sizes. The method can also handle χ2 distributed test statistics which enables power and sample size calculations for a much wider class of models, including high-dimensional generalized linear models which are used, e.g., for RNA-seq data analysis. The performance of the method is evaluated using simulated and experimental data from several microarray and next-generation sequencing experiments. Furthermore, we compare our proposed method for estimation of the density of effect sizes from pilot data with a recent proposed method specific for two group comparisons.