RESUMO
Determining the balance between DNA double strand break repair (DSBR) pathways is essential for understanding treatment response in cancer. We report a method for simultaneously measuring non-homologous end joining (NHEJ), homologous recombination (HR), and microhomology-mediated end joining (MMEJ). Using this method, we show that patient-derived glioblastoma (GBM) samples with acquired temozolomide (TMZ) resistance display elevated HR and MMEJ activity, suggesting that these pathways contribute to treatment resistance. We screen clinically relevant small molecules for DSBR inhibition with the aim of identifying improved GBM combination therapy regimens. We identify the ATM kinase inhibitor, AZD1390, as a potent dual HR/MMEJ inhibitor that suppresses radiation-induced phosphorylation of DSBR proteins, blocks DSB end resection, and enhances the cytotoxic effects of TMZ in treatment-naïve and treatment-resistant GBMs with TP53 mutation. We further show that a combination of G2/M checkpoint deficiency and reliance upon ATM-dependent DSBR renders TP53 mutant GBMs hypersensitive to TMZ/AZD1390 and radiation/AZD1390 combinations. This report identifies ATM-dependent HR and MMEJ as targetable resistance mechanisms in TP53-mutant GBM and establishes an approach for simultaneously measuring multiple DSBR pathways in treatment selection and oncology research.
Assuntos
Proteínas Mutadas de Ataxia Telangiectasia , Quebras de DNA de Cadeia Dupla , Glioblastoma , Temozolomida , Proteína Supressora de Tumor p53 , Humanos , Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , Proteínas Mutadas de Ataxia Telangiectasia/antagonistas & inibidores , Proteínas Mutadas de Ataxia Telangiectasia/genética , Glioblastoma/genética , Glioblastoma/tratamento farmacológico , Glioblastoma/metabolismo , Glioblastoma/patologia , Proteína Supressora de Tumor p53/metabolismo , Proteína Supressora de Tumor p53/genética , Quebras de DNA de Cadeia Dupla/efeitos dos fármacos , Temozolomida/farmacologia , Linhagem Celular Tumoral , Mutação , Resistencia a Medicamentos Antineoplásicos/genética , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Reparo do DNA/efeitos dos fármacos , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/tratamento farmacológico , Neoplasias Encefálicas/patologia , Neoplasias Encefálicas/metabolismo , Animais , Reparo do DNA por Junção de Extremidades/efeitos dos fármacos , Camundongos , Fosforilação/efeitos dos fármacosRESUMO
BACKGROUND: High-grade gliomas (HGG) are aggressive brain tumors associated with short median patient survival and limited response to therapies, driving the need to develop tools to improve patient outcomes. Patient-derived xenograft (PDX) models, such as mouse PDX, have emerged as potential Avatar platforms for personalized oncology approaches, but the difficulty for some human grafts to grow successfully and the long time required for mice to develop tumors preclude their use for HGG. METHODS: We used a rapid and efficient ex-ovo chicken embryo chorioallantoic membrane (CAM) culture system to evaluate the efficacy of oncologic drug options for HGG patients. RESULTS: Implantation of fresh glioma tissue fragments from 59 of 60 patients, that include difficult-to-grow IDH-mutated samples, successfully established CAM tumor xenografts within 7 days, with a tumor take rate of 98.3%. These xenografts faithfully recapitulate the histological and molecular characteristics of the primary tumor, and the ability of individual fragments to form tumors was predictive of poor patient prognosis. Treatment of drug-sensitive or drug-resistant xenografts indicates that the CAM-glioma assay enables testing tumor sensitivity to temozolomide and carboplatin at doses consistent with those administered to patients. In a proof-of-concept study involving 14 HGG patients, we observed a correlation of 100% between the CAM xenograft response to temozolomide or carboplatin and the clinical response of patients. CONCLUSION: The CAM-glioma model is a fast and reliable assay that has the potential to serve as a complementary model to drug discovery and a real-time Avatar platform to predict the best treatment for HGG patients.
Assuntos
Neoplasias Encefálicas , Glioma , Humanos , Embrião de Galinha , Camundongos , Animais , Temozolomida/farmacologia , Xenoenxertos , Carboplatina , Glioma/tratamento farmacológico , Neoplasias Encefálicas/tratamento farmacológico , Modelos Animais de Doenças , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
The epidermal growth factor receptor (EGFR) is a prime oncogene that is frequently amplified in glioblastomas. Here we demonstrate a new tumour-suppressive function of EGFR in EGFR-amplified glioblastomas regulated by EGFR ligands. Constitutive EGFR signalling promotes invasion via activation of a TAB1-TAK1-NF-κB-EMP1 pathway, resulting in large tumours and decreased survival in orthotopic models. Ligand-activated EGFR promotes proliferation and surprisingly suppresses invasion by upregulating BIN3, which inhibits a DOCK7-regulated Rho GTPase pathway, resulting in small hyperproliferating non-invasive tumours and improved survival. Data from The Cancer Genome Atlas reveal that in EGFR-amplified glioblastomas, a low level of EGFR ligands confers a worse prognosis, whereas a high level of EGFR ligands confers an improved prognosis. Thus, increased EGFR ligand levels shift the role of EGFR from oncogene to tumour suppressor in EGFR-amplified glioblastomas by suppressing invasion. The tumour-suppressive function of EGFR can be activated therapeutically using tofacitinib, which suppresses invasion by increasing EGFR ligand levels and upregulating BIN3.
Assuntos
Glioblastoma , Proteínas dos Microfilamentos/metabolismo , Linhagem Celular Tumoral , Receptores ErbB/genética , Receptores ErbB/metabolismo , Glioblastoma/metabolismo , Humanos , Ligantes , Oncogenes/genética , Regulação para CimaRESUMO
BACKGROUND: RBBP4 activates transcription by histone acetylation, but the partner histone acetyltransferases are unknown. Thus, we investigated the hypothesis that RBBP4 interacts with p300 in a complex in glioblastoma (GBM). METHODS: shRNA silencing of RBBP4 or p300 and RNAseq was used to identify genes co-regulated by RBBP4 and p300 in GBM43 patient-derived xenograft (PDX). RBBP4/p300 complex was demonstrated using proximity ligation assay (PLA) and ChIPseq delineated histone H3 acetylation and RBBP4/p300 complex binding in promoters/enhancers. Temozolomide (TMZ)-induced DNA double strand breaks (DSBs) were evaluated by γ-H2AX and proliferation by CyQuant and live cell monitoring assays. In vivo efficacy was based on survival of mice with orthotopic tumors. RESULTS: shRBBP4 and shp300 downregulated 4768 genes among which 1485 (31%) were commonly downregulated by both shRNAs, while upregulated genes were 2484, including 863 (35%) common genes. The pro-survival genes were the top-ranked among the downregulated genes, including C-MYC. RBBP4/p300 complex was demonstrated in the nucleus, and shRBBP4 or shp300 significantly sensitized GBM cells to TMZ compared to the control shNT in vitro (P < .05). Moreover, TMZ significantly prolonged the survival of mice bearing GBM22-shRBBP4 orthotopic tumors compared with control shNT tumors (median shNT survival 52 days vs. median shRBBP4 319 days; P = .001). CREB-binding protein (CBP)/p300 inhibitor CPI-1612 suppressed H3K27Ac and RBBP4/p300 complex target proteins, including C-MYC, and synergistically sensitized TMZ in vitro. Pharmacodynamic evaluation confirmed brain penetration by CPI-1612 supporting further investigation to evaluate efficacy to sensitize TMZ. CONCLUSIONS: RBBP4/p300 complex is present in GBM cells and is a potential therapeutic target.
Assuntos
Neoplasias Encefálicas , Proteína p300 Associada a E1A , Glioblastoma , Proteína 4 de Ligação ao Retinoblastoma , Acetilação , Animais , Neoplasias Encefálicas/tratamento farmacológico , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/patologia , Linhagem Celular Tumoral , Sobrevivência Celular , Resistencia a Medicamentos Antineoplásicos , Proteína p300 Associada a E1A/genética , Proteína p300 Associada a E1A/metabolismo , Glioblastoma/tratamento farmacológico , Glioblastoma/genética , Glioblastoma/metabolismo , Humanos , Camundongos , Regiões Promotoras Genéticas , Proteína 4 de Ligação ao Retinoblastoma/genética , Proteína 4 de Ligação ao Retinoblastoma/metabolismo , Temozolomida/farmacologia , Temozolomida/uso terapêutico , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Frontotemporal lobar degeneration with TDP-43 inclusions (FTLD-TDP) is a complex heterogeneous neurodegenerative disorder for which mechanisms are poorly understood. To explore transcriptional changes underlying FTLD-TDP, we performed RNA-sequencing on 66 genetically unexplained FTLD-TDP patients, 24 FTLD-TDP patients with GRN mutations and 24 control participants. Using principal component analysis, hierarchical clustering, differential expression and coexpression network analyses, we showed that GRN mutation carriers and FTLD-TDP-A patients without a known mutation shared a common transcriptional signature that is independent of GRN loss-of-function. After combining both groups, differential expression as compared to the control group and coexpression analyses revealed alteration of processes related to immune response, synaptic transmission, RNA metabolism, angiogenesis and vesicle-mediated transport. Deconvolution of the data highlighted strong cellular alterations that were similar in FTLD-TDP-A and GRN mutation carriers with NSF as a potentially important player in both groups. We propose several potentially druggable pathways such as the GABAergic, GDNF and sphingolipid pathways. Our findings underline new disease mechanisms and strongly suggest that affected pathways in GRN mutation carriers extend beyond GRN and contribute to genetically unexplained forms of FTLD-TDP-A.
Assuntos
Demência Frontotemporal , Degeneração Lobar Frontotemporal , Progranulinas , Encéfalo/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Demência Frontotemporal/genética , Demência Frontotemporal/metabolismo , Degeneração Lobar Frontotemporal/genética , Degeneração Lobar Frontotemporal/metabolismo , Humanos , Peptídeos e Proteínas de Sinalização Intercelular/genética , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Mutação , Progranulinas/genética , Progranulinas/metabolismo , TranscriptomaRESUMO
BACKGROUND: Antibody drug conjugates (ADCs) targeting the epidermal growth factor receptor (EGFR), such as depatuxizumab mafodotin (Depatux-M), is a promising therapeutic strategy for glioblastoma (GBM) but recent clinical trials did not demonstrate a survival benefit. Understanding the mechanisms of failure for this promising strategy is critically important. METHODS: PDX models were employed to study efficacy of systemic vs intracranial delivery of Depatux-M. Immunofluorescence and MALDI-MSI were performed to detect drug levels in the brain. EGFR levels and compensatory pathways were studied using quantitative flow cytometry, Western blots, RNAseq, FISH, and phosphoproteomics. RESULTS: Systemic delivery of Depatux-M was highly effective in nine of 10 EGFR-amplified heterotopic PDXs with survival extending beyond one year in eight PDXs. Acquired resistance in two PDXs (GBM12 and GBM46) was driven by suppression of EGFR expression or emergence of a novel short-variant of EGFR lacking the epitope for the Depatux-M antibody. In contrast to the profound benefit observed in heterotopic tumors, only two of seven intrinsically sensitive PDXs were responsive to Depatux-M as intracranial tumors. Poor efficacy in orthotopic PDXs was associated with limited and heterogeneous distribution of Depatux-M into tumor tissues, and artificial disruption of the BBB or bypass of the BBB by direct intracranial injection of Depatux-M into orthotopic tumors markedly enhanced the efficacy of drug treatment. CONCLUSIONS: Despite profound intrinsic sensitivity to Depatux-M, limited drug delivery into brain tumor may have been a key contributor to lack of efficacy in recently failed clinical trials.
Assuntos
Neoplasias Encefálicas , Glioblastoma , Imunoconjugados , Preparações Farmacêuticas , Anticorpos Monoclonais Humanizados , Barreira Hematoencefálica/metabolismo , Neoplasias Encefálicas/tratamento farmacológico , Linhagem Celular Tumoral , Receptores ErbB/genética , Receptores ErbB/metabolismo , Glioblastoma/tratamento farmacológico , HumanosRESUMO
BACKGROUND: Currently, there is no biologically based rationale for drug selection in migraine prophylactic treatment. METHODS: To investigate the genetic variation underlying treatment response to verapamil prophylaxis, we selected 225 patients from a longitudinally established, deeply phenotyped migraine database (N = 5983), and collected uninterrupted quantitated verapamil treatment response data and DNA for these 225 cases. We recorded the number of headache days in the four weeks preceding treatment with verapamil and for four weeks, following completion of a treatment period with verapamil lasting at least five weeks. Whole-exome sequencing (WES) was applied to a discovery cohort consisting of 21 definitive responders and 14 definitive non-responders, and the identified single nucleotide polymorphisms (SNPs) showing significant association were genotyped in a separate confirmation cohort (185 verapamil treated patients). Statistical analysis of the WES data from the discovery cohort identified 524 SNPs associated with verapamil responsiveness (p < 0.01); among them, 39 SNPs were validated in the confirmatory cohort (n = 185) which included the full range of response to verapamil from highly responsive to not responsive. RESULTS: Fourteen SNPs were confirmed by both percentage and arithmetic statistical approaches. Pathway and protein network analysis implicated myo-inositol biosynthetic and phospholipase-C second messenger pathways in verapamil responsiveness, emphasizing the earlier pathogenic understanding of migraine. No association was found between genetic variation in verapamil metabolic enzymes and treatment response. CONCLUSION: Our findings demonstrate that genetic analysis in well-characterized subpopulations can yield important pharmacogenetic information pertaining to the mechanism of anti-migraine prophylactic medications.
Assuntos
Transtornos de Enxaqueca/genética , Variantes Farmacogenômicos , Polimorfismo de Nucleotídeo Único , Vasodilatadores/uso terapêutico , Verapamil/uso terapêutico , Quimioprevenção , Humanos , Transtornos de Enxaqueca/tratamento farmacológico , Transtornos de Enxaqueca/prevenção & controle , Mapas de Interação de Proteínas , Fosfolipases Tipo C/genética , Fosfolipases Tipo C/metabolismo , Vasodilatadores/administração & dosagem , Verapamil/administração & dosagemRESUMO
T cell prolymphocytic leukemia (T-PLL) is a rare disease with aggressive clinical course. Cytogenetic analysis, whole-exome and whole-genome sequencing have identified primary structural alterations in T-PLL, including inversion, translocation and copy number variation. Recurrent somatic mutations were also identified in genes encoding chromatin regulators and those in the JAK-STAT signaling pathway. Epigenetic alterations are the hallmark of many cancers. However, genome-wide epigenomic profiles have not been reported in T-PLL, limiting the mechanistic study of its carcinogenesis. We hypothesize epigenetic mechanisms also play a key role in T-PLL pathogenesis. To systematically test this hypothesis, we generated genome-wide maps of regulatory regions using H3K4me3 and H3K27ac ChIP-seq, as well as RNA-seq data in both T-PLL patients and healthy individuals. We found that genes down-regulated in T-PLL are mainly associated with defense response, immune system or adaptive immune response, while up-regulated genes are enriched in developmental process, as well as WNT signaling pathway with crucial roles in cell fate decision. In particular, our analysis revealed a global alteration of regulatory landscape in T-PLL, with differential peaks highly enriched for binding motifs of immune related transcription factors, supporting the epigenetic regulation of oncogenes and genes involved in DNA damage response and T-cell activation. Together, our work reveals a causal role of epigenetic dysregulation in T-PLL.
Assuntos
Reprogramação Celular/genética , Epigênese Genética/genética , Epigênese Genética/fisiologia , Leucemia Prolinfocítica de Células T/genética , Leucemia Prolinfocítica de Células T/patologia , Transcrição Gênica/genética , Variações do Número de Cópias de DNA , Dano ao DNA/genética , Estudo de Associação Genômica Ampla , Humanos , Leucemia Prolinfocítica de Células T/imunologia , Ativação Linfocitária/genética , Linfócitos T/imunologia , Via de Sinalização Wnt/fisiologiaRESUMO
Chronic lymphocytic leukemia (CLL) is the most common adult leukemia in Western countries. It has a strong genetic basis, showing a ~ 8-fold increased risk of CLL in first-degree relatives. Genome-wide association studies (GWAS) have identified 41 risk variants across 41 loci. However, for a majority of the loci, the functional variants and the mechanisms underlying their causal roles remain undefined. Here, we examined the genetic and epigenetic features associated with 12 index variants, along with any correlated (r2 ≥ 0.5) variants, at the CLL risk loci located outside of gene promoters. Based on publicly available ChIP-seq and chromatin accessibility data as well as our own ChIP-seq data from CLL patients, we identified six candidate functional variants at six loci and at least two candidate functional variants at each of the remaining six loci. The functional variants are predominantly located within enhancers or super-enhancers, including bi-directionally transcribed enhancers, which are often restricted to immune cell types. Furthermore, we found that, at 78% of the functional variants, the alternative alleles altered the transcription factor binding motifs or histone modifications, indicating the involvement of these variants in the change of local chromatin state. Finally, the enhancers carrying functional variants physically interacted with genes enriched in the type I interferon signaling pathway, apoptosis, or TP53 network that are known to play key roles in CLL. These results support the regulatory roles for inherited noncoding variants in the pathogenesis of CLL.
Assuntos
Elementos Facilitadores Genéticos/genética , Predisposição Genética para Doença , Estudo de Associação Genômica Ampla , Leucemia Linfocítica Crônica de Células B/genética , Alelos , Cromatina/genética , Epigênese Genética/genética , Feminino , Humanos , Leucemia Linfocítica Crônica de Células B/patologia , Masculino , Polimorfismo de Nucleotídeo Único/genética , Ligação Proteica , Fatores de Risco , Proteína Supressora de Tumor p53/genéticaRESUMO
Tumors acquire numerous mutations during development and progression. When translated into proteins, these mutations give rise to neoantigens that can be recognized by T cells and generate antibodies, representing an exciting direction of cancer immunotherapy. While neoantigens have been reported in many cancer types, the profiling of neoantigens often focused on the class-I subtype that are presented to CD8 + T cells, and the relationship between neoantigen load and clinical outcomes was often inconsistent among cancer types. In this study, we described an informatics workflow, REAL-neo, for identification, quality control (QC), and prioritization of both class-I and class-II human leukocyte antigen (HLA) bound neoantigens that arise from somatic single nucleotide mutations (SNM), small insertions and deletions (INDEL), and gene fusions. We applied REAL-neo to 835 primary breast tumors in the Cancer Genome Atlas (TCGA) and performed comprehensive profiling and characterization of the detected neoantigens. We found recurrent HLA class-I and class-II restricted neoantigens across breast cancer cases, and uncovered associations between neoantigen load and clinical traits. Both class-I and class-II neoantigen loads from SNM and INDEL were found to predict overall survival independent of tumor mutational burden (TMB), breast cancer subtypes, tumor-infiltrating lymphocyte (TIL) levels, tumor stage, and age at diagnosis. Our study highlighted the importance of accurate and comprehensive neoantigen profiling and QC, and is the first to report the predictive value of neoantigen load for overall survival in breast cancer.
Assuntos
Antígenos de Neoplasias , Neoplasias da Mama , Antígenos de Neoplasias/genética , Neoplasias da Mama/diagnóstico , Neoplasias da Mama/genética , Feminino , Antígenos de Histocompatibilidade Classe I , Antígenos de Histocompatibilidade Classe II , Humanos , Imunoterapia , Linfócitos do Interstício Tumoral , Taxa de SobrevidaRESUMO
Seventy-six FDA-approved oncology drugs and emerging therapeutics were evaluated in 25 multiple myeloma (MM) and 15 non-Hodgkin's lymphoma cell lines and in 113 primary MM samples. Ex vivo drug sensitivities were mined for associations with clinical phenotype, cytogenetic, genetic mutation, and transcriptional profiles. In primary MM samples, proteasome inhibitors, dinaciclib, selinexor, venetoclax, auranofin, and histone deacetylating agents had the broadest cytotoxicity. Of interest, newly diagnosed patient samples were globally less sensitive especially to bromodomain inhibitors, inhibitors of receptor tyrosine kinases or non-receptor kinases, and DNA synthesis inhibitors. Clustering demonstrated six broad groupings of drug sensitivity linked with genomic biomarkers and clinical outcomes. For example, our findings mimic clinical observations of increased venetoclax responsiveness in t(11;14) patients but also identify an increased sensitivity profile in untreated patients, standard genetic risk, low plasma cell S-Phase, and in the absence of Gain(1q) and t(4;14). In contrast, increased ex vivo responsiveness to selinexor was associated with biomarkers of poor prognosis and later relapse patients. This "direct to drug" screening resource, paired with functional genomics, has the potential to successfully direct appropriate individualized therapeutic approaches in MM and to enrich clinical trials for likely responders.
Assuntos
Antineoplásicos/farmacologia , Ensaios de Seleção de Medicamentos Antitumorais/métodos , Mieloma Múltiplo/tratamento farmacológico , Compostos Bicíclicos Heterocíclicos com Pontes/farmacologia , Linhagem Celular Tumoral , Humanos , Hidrazinas/farmacologia , Mieloma Múltiplo/genética , Medicina de Precisão/métodos , Sulfonamidas/farmacologia , Triazóis/farmacologia , Células Tumorais CultivadasRESUMO
Next-generation sequencing identified about 60 genes recurrently mutated in chronic lymphocytic leukemia (CLL). We examined the additive prognostic value of the total number of recurrently mutated CLL genes (i.e., tumor mutational load [TML]) or the individually mutated genes beyond the CLL international prognostic index (CLL-IPI) in newly diagnosed CLL and high-count monoclonal B-cell lymphocytosis (HC MBL). We sequenced 59 genes among 557 individuals (112 HC MBL/445 CLL) in a multi-stage design, to estimate hazard ratios (HR) and 95% confidence intervals (CI) for time-to-first treatment (TTT), adjusted for CLL-IPI and sex. TML was associated with shorter TTT in the discovery and validation cohorts, with a combined estimate of continuous HR = 1.27 (CI:1.17-1.39, P = 2.6 × 10-8 ; c-statistic = 0.76). When stratified by CLL-IPI, the association of TML with TTT was stronger and validated within low/intermediate risk (combined HR = 1.54, CI:1.37-1.72, P = 7.0 × 10-14 ). Overall, 80% of low/intermediate CLL-IPI cases with two or more mutated genes progressed to require therapy within 5 years, compared to 24% among those without mutations. TML was also associated with shorter TTT in the HC MBL cohort (HR = 1.53, CI:1.12-2.07, P = .007; c-statistic = 0.71). TML is a strong prognostic factor for TTT independent of CLL-IPI, especially among low/intermediate CLL-IPI risk, and a better predictor than any single gene. Mutational screening at early stages may improve risk stratification and better predict TTT.
Assuntos
Sequenciamento de Nucleotídeos em Larga Escala/métodos , Leucemia Linfocítica Crônica de Células B/genética , Linfocitose/metabolismo , Adulto , Idoso , Idoso de 80 Anos ou mais , Feminino , Humanos , Leucemia Linfocítica Crônica de Células B/patologia , Masculino , Pessoa de Meia-Idade , PrognósticoRESUMO
PURPOSE: Glioblastoma is the most frequent and lethal primary brain tumor. Development of novel therapies relies on the availability of relevant preclinical models. We have established a panel of 96 glioblastoma patient-derived xenografts (PDX) and undertaken its genomic and phenotypic characterization. EXPERIMENTAL DESIGN: PDXs were established from glioblastoma, IDH-wildtype (n = 93), glioblastoma, IDH-mutant (n = 2), diffuse midline glioma, H3 K27M-mutant (n = 1), and both primary (n = 60) and recurrent (n = 34) tumors. Tumor growth rates, histopathology, and treatment response were characterized. Integrated molecular profiling was performed by whole-exome sequencing (WES, n = 83), RNA-sequencing (n = 68), and genome-wide methylation profiling (n = 76). WES data from 24 patient tumors was compared with derivative models. RESULTS: PDXs recapitulate many key phenotypic and molecular features of patient tumors. Orthotopic PDXs show characteristic tumor morphology and invasion patterns, but largely lack microvascular proliferation and necrosis. PDXs capture common and rare molecular drivers, including alterations of TERT, EGFR, PTEN, TP53, BRAF, and IDH1, most at frequencies comparable with human glioblastoma. However, PDGFRA amplification was absent. RNA-sequencing and genome-wide methylation profiling demonstrated broad representation of glioblastoma molecular subtypes. MGMT promoter methylation correlated with increased survival in response to temozolomide. WES of 24 matched patient tumors showed preservation of most genetic driver alterations, including EGFR amplification. However, in four patient-PDX pairs, driver alterations were gained or lost on engraftment, consistent with clonal selection. CONCLUSIONS: Our PDX panel captures the molecular heterogeneity of glioblastoma and recapitulates many salient genetic and phenotypic features. All models and genomic data are openly available to investigators.
Assuntos
Biomarcadores Tumorais/genética , Sequenciamento do Exoma/métodos , Genótipo , Glioblastoma/classificação , Glioblastoma/genética , Mutação , Fenótipo , Adulto , Idoso , Idoso de 80 Anos ou mais , Animais , Antineoplásicos Alquilantes/farmacologia , Neoplasias Encefálicas/classificação , Neoplasias Encefálicas/tratamento farmacológico , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/patologia , Metilação de DNA , Metilases de Modificação do DNA/genética , Enzimas Reparadoras do DNA/genética , Receptores ErbB/genética , Feminino , Glioblastoma/tratamento farmacológico , Glioblastoma/patologia , Humanos , Isocitrato Desidrogenase/genética , Masculino , Camundongos , Pessoa de Meia-Idade , Estadiamento de Neoplasias , Regiões Promotoras Genéticas , Taxa de Sobrevida , Temozolomida/farmacologia , Proteínas Supressoras de Tumor/genética , Ensaios Antitumorais Modelo de Xenoenxerto , Adulto JovemRESUMO
PURPOSE: The purpose of the study was to develop and evaluate an automated digitization algorithm for high-dose-rate cervix brachytherapy, with the goal of reducing the duration of treatment planning, staff resources, variability, and potential for human error. METHODS: An automated digitization algorithm was developed and retrospectively evaluated using treatment planning data from 10 patients with cervix cancer who were treated with a titanium tandem and ovoids applicator set. Applicators were segmented, without human interaction, by thresholding CT images to isolate high-density voxels and assigning the voxels to applicator and nonapplicator structures using HDBSCAN, a density-based linkage clustering algorithm. The applicator contours were determined from the centroid of the clustered voxels on each image slice and written to a treatment plan file. Automated contours were evaluated against manual digitization using distance and dosimetric metrics. RESULTS: A close agreement between automatic and manual digitization was observed. The mean magnitude of contour disagreement for 10 patients equaled 0.3 mm. Hausdorff distances were ≤1.0 mm. The applicator tip coordinates had submillimeter agreement. The median and mean dose volume histogram parameter differences were less than or equal to 1% for high-risk clinical target volume D90, high-risk clinical target volume D95, bladder D2cc, rectum D2cc, large bowel D2cc, and small bowel D2cc. The average execution time for the automated algorithm was less than 30 s. CONCLUSION: The digitization of titanium tandem and ovoids applicators for high-dose-rate brachytherapy treatment planning can be automated using straightforward thresholding and clustering algorithms. The adoption of automated digitization is expected to improve the consistency of treatment plans and reduce the duration of treatment planning.
Assuntos
Algoritmos , Braquiterapia/métodos , Planejamento da Radioterapia Assistida por Computador/métodos , Neoplasias do Colo do Útero/radioterapia , Automação , Feminino , Humanos , Intestinos , Órgãos em Risco , Doses de Radiação , Dosagem Radioterapêutica , Estudos Retrospectivos , Tomografia Computadorizada por Raios X , Bexiga Urinária , Neoplasias do Colo do Útero/diagnóstico por imagemRESUMO
Anaplastic large cell lymphomas (ALCLs) represent a relatively common group of T-cell non-Hodgkin lymphomas (T-NHLs) that are unified by similar pathologic features but demonstrate marked genetic heterogeneity. ALCLs are broadly classified as being anaplastic lymphoma kinase (ALK)+ or ALK-, based on the presence or absence of ALK rearrangements. Exome sequencing of 62 T-NHLs identified a previously unreported recurrent mutation in the musculin gene, MSC E116K, exclusively in ALK- ALCLs. Additional sequencing for a total of 238 T-NHLs confirmed the specificity of MSC E116K for ALK- ALCL and further demonstrated that 14 of 15 mutated cases (93%) had coexisting DUSP22 rearrangements. Musculin is a basic helix-loop-helix (bHLH) transcription factor that heterodimerizes with other bHLH proteins to regulate lymphocyte development. The E116K mutation localized to the DNA binding domain of musculin and permitted formation of musculin-bHLH heterodimers but prevented their binding to authentic target sequence. Functional analysis showed MSCE116K acted in a dominant-negative fashion, reversing wild-type musculin-induced repression of MYC and cell cycle inhibition. Chromatin immunoprecipitation-sequencing and transcriptome analysis identified the cell cycle regulatory gene E2F2 as a direct transcriptional target of musculin. MSCE116K reversed E2F2-induced cell cycle arrest and promoted expression of the CD30-IRF4-MYC axis, whereas its expression was reciprocally induced by binding of IRF4 to the MSC promoter. Finally, ALCL cells expressing MSC E116K were preferentially targeted by the BET inhibitor JQ1. These findings identify a novel recurrent MSC mutation as a key driver of the CD30-IRF4-MYC axis and cell cycle progression in a unique subset of ALCLs.
Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Linfoma Anaplásico de Células Grandes/genética , Quinase do Linfoma Anaplásico/genética , Ciclo Celular/genética , Regulação Neoplásica da Expressão Gênica/genética , Humanos , MutaçãoRESUMO
Chromatin immunoprecipitation and sequencing (ChIP-seq) has been widely used to map DNA-binding proteins, histone proteins and their modifications. ChIP-seq data contains redundant reads termed duplicates, referring to those mapping to the same genomic location and strand. There are two main sources of duplicates: polymerase chain reaction (PCR) duplicates and natural duplicates. Unlike natural duplicates that represent true signals from sequencing of independent DNA templates, PCR duplicates are artifacts originating from sequencing of identical copies amplified from the same DNA template. In analysis, duplicates are removed from peak calling and signal quantification. Nevertheless, a significant portion of the duplicates is believed to represent true signals. Obviously, removing all duplicates will underestimate the signal level in peaks and impact the identification of signal changes across samples. Therefore, an in-depth evaluation of the impact from duplicate removal is needed. Using eight public ChIP-seq datasets from three narrow-peak and two broad-peak marks, we tried to understand the distribution of duplicates in the genome, the extent by which duplicate removal impacts peak calling and signal estimation, and the factors associated with duplicate level in peaks. The three PCR-free histone H3 lysine 4 trimethylation (H3K4me3) ChIP-seq data had about 40% duplicates and 97% of them were within peaks. For the other datasets generated with PCR amplification of ChIP DNA, as expected, the narrow-peak marks have a much higher proportion of duplicates than the broad-peak marks. We found that duplicates are enriched in peaks and largely represent true signals, more conspicuous in those with high confidence. Furthermore, duplicate level in peaks is strongly correlated with the target enrichment level estimated using nonredundant reads, which provides the basis to properly allocate duplicates between noise and signal. Our analysis supports the feasibility of retaining the portion of signal duplicates into downstream analysis, thus alleviating the limitation of complete deduplication.