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1.
Int J Mol Sci ; 22(9)2021 Apr 29.
Artículo en Inglés | MEDLINE | ID: mdl-33946969

RESUMEN

The cytogenetic and molecular assessment of deletions, amplifications and rearrangements are key aspects in the diagnosis and therapy of cancer. Not only the initial evaluation and classification of the disease, but also the follow-up of the tumor rely on these laboratory approaches. The therapeutic choice can be guided by the results of the laboratory testing. Genetic deletions and/or amplifications directly affect the susceptibility or the resistance to specific therapies. In an era of personalized medicine, the correct and reliable molecular characterization of the disease, also during the therapeutic path, acquires a pivotal role. Molecular assays like multiplex ligation-dependent probe amplification and droplet digital PCR represent exceptional tools for a sensitive and reliable detection of genetic alterations and deserve a role in molecular oncology. In this manuscript we provide a technical comparison of these two approaches with the golden standard represented by fluorescence in situ hybridization. We also describe some relevant targets currently evaluated with these techniques in solid and hematologic tumors.


Asunto(s)
Variaciones en el Número de Copia de ADN , ADN de Neoplasias/genética , Tecnología Digital/métodos , Reordenamiento Génico , Proteínas de Neoplasias/genética , Neoplasias/genética , Reacción en Cadena de la Polimerasa/métodos , Aberraciones Cromosómicas , Emulsiones , Determinación de Punto Final/métodos , Fluorometría , Humanos , Hibridación Fluorescente in Situ/métodos , Reacción en Cadena de la Polimerasa Multiplex/métodos , Proteínas de Fusión Oncogénica/genética , Sensibilidad y Especificidad
2.
Nat Commun ; 12(1): 2204, 2021 04 13.
Artículo en Inglés | MEDLINE | ID: mdl-33850139

RESUMEN

Intra-tumor heterogeneity renders the identification of somatic single-nucleotide variants (SNVs) a challenging problem. In particular, low-frequency SNVs are hard to distinguish from sequencing artifacts. While the increasing availability of multi-sample tumor DNA sequencing data holds the potential for more accurate variant calling, there is a lack of high-sensitivity multi-sample SNV callers that utilize these data. Here we report Moss, a method to identify low-frequency SNVs that recur in multiple sequencing samples from the same tumor. Moss provides any existing single-sample SNV caller the ability to support multiple samples with little additional time overhead. We demonstrate that Moss improves recall while maintaining high precision in a simulated dataset. On multi-sample hepatocellular carcinoma, acute myeloid leukemia and colorectal cancer datasets, Moss identifies new low-frequency variants that meet manual review criteria and are consistent with the tumor's mutational signature profile. In addition, Moss detects the presence of variants in more samples of the same tumor than reported by the single-sample caller. Moss' improved sensitivity in SNV calling will enable more detailed downstream analyses in cancer genomics.


Asunto(s)
ADN de Neoplasias/genética , Neoplasias Hepáticas/genética , Nucleótidos , Algoritmos , Carcinoma Hepatocelular , Neoplasias Colorrectales/genética , Frecuencia de los Genes , Genómica/métodos , Humanos , Leucemia Mieloide Aguda/genética , Mutación , Polimorfismo de Nucleótido Simple
3.
Int J Mol Sci ; 22(8)2021 Apr 11.
Artículo en Inglés | MEDLINE | ID: mdl-33920410

RESUMEN

Downregulation of multiple tumor suppressor genes (TSGs) plays an important role in cancer formation. Recent evidence has accumulated that cancer progression involves genome-wide alteration of epigenetic modifications, which may cause downregulation of the tumor suppressor gene. Using hepatocellular carcinoma (HCC) as a system, we mapped 5-methylcytosine signal at a genome-wide scale using nanopore sequencing technology to identify novel TSGs. Integration of methylation data with gene transcription profile of regenerated liver and primary HCCs allowed us to identify 10 potential tumor suppressor gene candidates. Subsequent validation led us to focus on functionally characterizing one candidate-glucokinase (GCK). We show here that overexpression of GCK inhibits the proliferation of HCC cells via induction of intracellular lactate accumulation and subsequently causes energy crisis due to NAD+ depletion. This suggests GCK functions as a tumor suppressor gene and may be involved in HCC development. In conclusion, these data provide valuable clues for further investigations of the process of tumorigenesis in human cancer.


Asunto(s)
Carcinoma Hepatocelular , Metilación de ADN , ADN de Neoplasias , Genes Supresores de Tumor , Neoplasias Hepáticas , Secuenciación de Nanoporos , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/metabolismo , ADN de Neoplasias/genética , ADN de Neoplasias/metabolismo , Estudio de Asociación del Genoma Completo , Células Hep G2 , Humanos , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/metabolismo
4.
Molecules ; 26(9)2021 Apr 25.
Artículo en Inglés | MEDLINE | ID: mdl-33922925

RESUMEN

Exosomes are the small vesicles that are secreted by different types of normal and tumour cells and can incorporate and transfer their cargo to the recipient cells. The main goal of the present work was to study the tumour exosomes' ability to accumulate the parent mutant DNA or RNA transcripts with their following transfer to the surrounding cells. The experiments were performed on the MCF7 breast cancer cells that are characterized by the unique coding mutation in the PIK3CA gene. Using two independent methods, Sanger sequencing and allele-specific real-time PCR, we revealed the presence of the fragments of the mutant DNA and RNA transcripts in the exosomes secreted by the MCF7 cells. Furthermore, we demonstrated the MCF7 exosomes' ability to incorporate into the heterologous MDA-MB-231 breast cancer cells supporting the possible transferring of the exosomal cargo into the recipient cells. Sanger sequencing of the DNA from MDA-MB-231 cells (originally bearing a wild type of PIK3CA) treated with MCF7 exosomes showed no detectable amount of mutant DNA or RNA; however, using allele-specific real-time PCR, we revealed a minor signal from amplification of a mutant allele, showing a slight increase of mutant DNA in the exosome-treated MDA-MB-231 cells. The results demonstrate the exosome-mediated secretion of the fragments of mutant DNA and mRNA by the cancer cells and the exosomes' ability to transfer their cargo into the heterologous cells.


Asunto(s)
Neoplasias de la Mama/genética , Fosfatidilinositol 3-Quinasa Clase I/genética , ADN de Neoplasias/genética , Exosomas/genética , Alelos , Neoplasias de la Mama/patología , Femenino , Humanos , Células MCF-7 , Mutación/genética , ARN Mensajero/genética
5.
Int J Mol Sci ; 22(6)2021 Mar 11.
Artículo en Inglés | MEDLINE | ID: mdl-33799581

RESUMEN

A palindrome in DNA consists of two closely spaced or adjacent inverted repeats. Certain palindromes have important biological functions as parts of various cis-acting elements and protein binding sites. However, many palindromes are known as fragile sites in the genome, sites prone to chromosome breakage which can lead to various genetic rearrangements or even cell death. The ability of certain palindromes to initiate genetic recombination lies in their ability to form secondary structures in DNA which can cause replication stalling and double-strand breaks. Given their recombinogenic nature, it is not surprising that palindromes in the human genome are involved in genetic rearrangements in cancer cells as well as other known recurrent translocations and deletions associated with certain syndromes in humans. Here, we bring an overview of current understanding and knowledge on molecular mechanisms of palindrome recombinogenicity and discuss possible implications of DNA palindromes in carcinogenesis. Furthermore, we overview the data on known palindromic sequences in the human genome and efforts to estimate their number and distribution, as well as underlying mechanisms of genetic rearrangements specific palindromic sequences cause.


Asunto(s)
Carcinogénesis/genética , ADN de Neoplasias/genética , Secuencias Invertidas Repetidas , Neoplasias/genética , Recombinación Genética , Translocación Genética , Secuencia de Bases , Carcinogénesis/metabolismo , Carcinogénesis/patología , Biología Computacional/métodos , Replicación del ADN , ADN de Neoplasias/química , ADN de Neoplasias/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Genoma Humano , Inestabilidad Genómica , Humanos , Neoplasias/metabolismo , Neoplasias/patología , Conformación de Ácido Nucleico , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo
6.
Science ; 372(6538)2021 04 09.
Artículo en Inglés | MEDLINE | ID: mdl-33833097

RESUMEN

Liquid biopsies that analyze cell-free DNA in blood plasma are used for noninvasive prenatal testing, oncology, and monitoring of organ transplant recipients. DNA molecules are released into the plasma from various bodily tissues. Physical and molecular features of cell-free DNA fragments and their distribution over the genome bear information about their tissues of origin. Moreover, patterns of DNA methylation of these molecules reflect those of their tissue sources. The nucleosomal organization and nuclease content of the tissue of origin affect the fragmentation profile of plasma DNA molecules, such as fragment size and end motifs. Besides double-stranded linear fragments, other topological forms of cell-free DNA also exist-namely circular and single-stranded molecules. Enhanced by these features, liquid biopsies hold promise for the noninvasive detection of tissue-specific pathologies with a range of clinical applications.


Asunto(s)
Ácidos Nucleicos Libres de Células/sangre , Ácidos Nucleicos Libres de Células/genética , Fragmentación del ADN , Metilación de ADN , ADN/sangre , ADN/genética , Biopsia Líquida , Animales , Biomarcadores/sangre , ADN Circular/sangre , ADN Mitocondrial/sangre , ADN de Neoplasias/sangre , ADN de Neoplasias/genética , Desoxirribonucleasas/metabolismo , Epigénesis Genética , Femenino , Feto , Humanos , Embarazo , Trasplantes
7.
Nat Metab ; 3(4): 558-570, 2021 04.
Artículo en Inglés | MEDLINE | ID: mdl-33833465

RESUMEN

Mitochondrial DNA (mtDNA) encodes protein subunits and translational machinery required for oxidative phosphorylation (OXPHOS). Using repurposed whole-exome sequencing data, in the present study we demonstrate that pathogenic mtDNA mutations arise in tumours at a rate comparable to those in the most common cancer driver genes. We identify OXPHOS complexes as critical determinants shaping somatic mtDNA mutation patterns across tumour lineages. Loss-of-function mutations accumulate at an elevated rate specifically in complex I and often arise at specific homopolymeric hotspots. In contrast, complex V is depleted of all non-synonymous mutations, suggesting that impairment of ATP synthesis and mitochondrial membrane potential dissipation are under negative selection. Common truncating mutations and rarer missense alleles are both associated with a pan-lineage transcriptional programme, even in cancer types where mtDNA mutations are comparatively rare. Pathogenic mutations of mtDNA are associated with substantial increases in overall survival of colorectal cancer patients, demonstrating a clear functional relationship between genotype and phenotype. The mitochondrial genome is therefore frequently and functionally disrupted across many cancers, with major implications for patient stratification, prognosis and therapeutic development.


Asunto(s)
Linaje de la Célula/genética , ADN Mitocondrial/genética , ADN de Neoplasias/genética , Fosforilación Oxidativa , Adenosina Trifosfato/biosíntesis , Neoplasias Colorrectales/genética , Exoma/genética , Genoma Humano/genética , Genoma Mitocondrial , Genotipo , Humanos , Potencial de la Membrana Mitocondrial/fisiología , Mitocondrias/genética , Mutación/genética , Fenotipo , ARN/genética
8.
Talanta ; 228: 122220, 2021 Jun 01.
Artículo en Inglés | MEDLINE | ID: mdl-33773726

RESUMEN

Tumor is a kind of abnormal organism generated by the proliferation and differentiation of cells in the body under the action of various initiating and promoting factors, which seriously threatens human life and health. Tumorigenesis is a gradual process that involves multistage reactions and the accumulation of mutations. Gene mutation usually occurs during tumorigenesis, and can be used for tumor diagnosis. Early diagnosis is the most effective way to improve the cure rate and reduce the mortality rate. Among the peripheral blood circulating tumor DNA (ctDNA), gene mutation in keeping with tumor cells can be detected, which can potentially replace tumor tissue section for early diagnosis. It has been considered as a liquid biopsy marker with good clinical application prospect. However, the high fragmentation and low concentration of ctDNA in blood result in the difficulty of tumor stage determination. Therefore, high sensitive and specific mutation detection methods have been developed to detect trace mutant ctDNA. At present, the approaches include digital PCR (dPCR), Bead, Emulsion, Amplification and Magnetic (BEAMing), Next Generation Sequencing (NGS), Amplification Refractory Mutation System (ARMS), etc. In this paper, the principle, characteristics, latest progress and application prospects of these methods are reviewed, which will facilitate researchers to choose appropriate ctDNA detection approaches.


Asunto(s)
ADN Tumoral Circulante , Neoplasias , Biomarcadores de Tumor/genética , ADN Tumoral Circulante/genética , ADN de Neoplasias/genética , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Biopsia Líquida , Mutación , Neoplasias/diagnóstico , Neoplasias/genética
9.
Medicine (Baltimore) ; 100(9): e24920, 2021 Mar 05.
Artículo en Inglés | MEDLINE | ID: mdl-33655954

RESUMEN

BACKGROUND: Previous studies have investigated the prognostic role of programmed death ligand 1 (PD-L1) expression in patients with neuroblastoma, while the results are still controversial. Therefore, we conducted a meta-analysis to clarify the relationship between the expression of PD-L1 and the prognosis of neuroblastoma. METHODS: Search electronic databases include PubMed, Cochrane, Embase, Scopus and Web of Science, and the search time is set to build the database until January 2021. Hazard ratio (HR) and 95% confidence interval (CI) were used to analyze the included results. Meta-analysis was performed using Stata 15.0 software. RESULTS: This review will be disseminated in print by peer-review. CONCLUSION: The study will provide updated evidence for the evaluation of whether the expression of PD-L1 is associated with poor prognosis in patients with neuroblastoma. ETHICS AND DISSEMINATION: The private information from individuals will not be published. This systematic review also should not damage participants' rights. Ethical approval is not available. The results may be published in a peer-reviewed journal or disseminated in relevant conferences. OSF REGISTRATION NUMBER: DOI 10.17605/OSF.IO/FBCY6.


Asunto(s)
Antígeno B7-H1/genética , ADN de Neoplasias/genética , Regulación Neoplásica de la Expresión Génica , Neuroblastoma/genética , Antígeno B7-H1/biosíntesis , Muerte Celular/genética , Supervivencia Celular , Humanos , Neuroblastoma/metabolismo , Neuroblastoma/patología , Pronóstico
10.
Medicine (Baltimore) ; 100(9): e24953, 2021 Mar 05.
Artículo en Inglés | MEDLINE | ID: mdl-33655961

RESUMEN

ABSTRACT: Colon cancer is one of the most common cancers in the world. To identify the candidate genes in the carcinogenesis and progression of colon cancer, the microarray datasets GSE10950, GSE44861 and GSE74602 were downloaded from the Gene Expression Omnibus (GEO) database. The differentially expressed genes (DEGs) were identified, and functional enrichment analyses were performed. A total of 176 DEGs were identified, consisting of 55 genes upregulated and 121 genes downregulated in colon cancer tissues compared to non-cancerous tissues. The DEGs were mainly enriched in mineral absorption, nitrogen metabolism and complement and coagulation cascades. By using STRING database analysis, we constructed a coexpression network composed of 140 nodes and 280 edges for the DEGs with a combined score >0.4 and a significant interaction relation. Thirteen hub genes were identified, and poor OS of patients was only associated with high expression of Matrix Metallopeptidase 7 (MMP7), which may be involved in the carcinogenesis, invasion or recurrence of colon cancer. In conclusion, we propose that the DEGs and hub genes identified in the present study may be regarded as diagnostic biomarkers for colon cancer. Moreover, the overexpression of MMP7 may correlate with poor prognosis.


Asunto(s)
Neoplasias del Colon/genética , Biología Computacional/métodos , Regulación Neoplásica de la Expresión Génica , Metaloproteinasa 7 de la Matriz/genética , Biomarcadores de Tumor/biosíntesis , Biomarcadores de Tumor/genética , Neoplasias del Colon/diagnóstico , Neoplasias del Colon/metabolismo , ADN de Neoplasias/genética , Bases de Datos Genéticas , Progresión de la Enfermedad , Perfilación de la Expresión Génica/métodos , Humanos , Metaloproteinasa 7 de la Matriz/biosíntesis , Análisis por Micromatrices , Pronóstico
11.
BMC Cancer ; 21(1): 207, 2021 Mar 01.
Artículo en Inglés | MEDLINE | ID: mdl-33648461

RESUMEN

BACKGROUND: Colorectal cancer (CRC) is one of the most common cancers worldwide; it is the fourth leading cause of death in the world and the third in Brazil. Mutations in the APC, DCC, KRAS and TP53 genes have been associated with the progression of sporadic CRC, occurring at defined pathological stages of the tumor progression and consequently modulating several genes in the corresponding signaling pathways. Therefore, the identification of gene signatures that occur at each stage during the CRC progression is critical and can present an impact on the diagnosis and prognosis of the patient. In this study, our main goal was to determine these signatures, by evaluating the gene expression of paired colorectal adenoma and adenocarcinoma samples to identify novel genetic markers in association to the adenoma-adenocarcinoma stage transition. METHODS: Ten paired adenoma and adenocarcinoma colorectal samples were subjected to microarray gene expression analysis. In addition, mutations in APC, KRAS and TP53 genes were investigated by DNA sequencing in paired samples of adenoma, adenocarcinoma, normal tissue, and peripheral blood from ten patients. RESULTS: Gene expression analysis revealed a signature of 689 differentially expressed genes (DEG) (fold-change> 2, p< 0.05), between the adenoma and adenocarcinoma paired samples analyzed. Gene pathway analysis using the 689 DEG identified important cancer pathways such as remodeling of the extracellular matrix and epithelial-mesenchymal transition. Among these DEG, the ETV4 stood out as one of the most expressed in the adenocarcinoma samples, further confirmed in the adenocarcinoma set of samples from the TCGA database. Subsequent in vitro siRNA assays against ETV4 resulted in the decrease of cell proliferation, colony formation and cell migration in the HT29 and SW480 colorectal cell lines. DNA sequencing analysis revealed KRAS and TP53 gene pathogenic mutations, exclusively in the adenocarcinomas samples. CONCLUSION: Our study identified a set of genes with high potential to be used as biomarkers in CRC, with a special emphasis on the ETV4 gene, which demonstrated involvement in proliferation and migration.


Asunto(s)
Adenocarcinoma/genética , Adenoma/genética , Neoplasias Colorrectales/genética , Genes Relacionados con las Neoplasias , Proteínas de Neoplasias/fisiología , Proteínas Proto-Oncogénicas c-ets/fisiología , Adenocarcinoma/química , Adenocarcinoma/patología , Adenoma/química , Adenoma/patología , Anciano , Biomarcadores de Tumor/genética , Brasil , División Celular/genética , Línea Celular Tumoral , Movimiento Celular/genética , Transformación Celular Neoplásica/genética , Neoplasias Colorrectales/química , Neoplasias Colorrectales/patología , ADN de Neoplasias/genética , Progresión de la Enfermedad , Femenino , Regulación Neoplásica de la Expresión Génica , Ontología de Genes , Humanos , Masculino , Persona de Mediana Edad , Proteínas de Neoplasias/antagonistas & inhibidores , Proteínas de Neoplasias/genética , Proteínas Proto-Oncogénicas c-ets/antagonistas & inhibidores , Proteínas Proto-Oncogénicas c-ets/genética , Interferencia de ARN , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/farmacología , Análisis de Matrices Tisulares , Transcriptoma , Ensayo de Tumor de Célula Madre
12.
Bull Cancer ; 108(4): 385-398, 2021 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-33685627

RESUMEN

Numerous epigenetic alterations are observed in cancer cells, and dysregulation of mono-ubiquitination of histone H2B (H2Bub1) has often been linked to tumorigenesis. H2Bub1 is a dynamic post-translational histone modification associated with transcriptional elongation and DNA damage response. Histone H2B monoubiquitination occurs in the site of lysine 120, written predominantly by E3 ubiquitin ligases RNF20/RNF40 and deubiquitinated by ubiquitin specific peptidase 22 (USP22). RNF20/40 is often altered in the primary tumors including colorectal cancer, breast cancer, ovarian cancer, prostate cancer, and lung cancer, and the loss of H2Bub1 is usually associated with poor prognosis in tumor patients. The purpose of this review is to summarize the current knowledge of H2Bub1 in transcription, DNA damage response and primary tumors. This review also provides novel options for exploiting the potential therapeutic target H2Bub1 in personalized cancer therapy.


Asunto(s)
Regulación Neoplásica de la Expresión Génica , Histonas/fisiología , Proteínas de Neoplasias/fisiología , Neoplasias/metabolismo , Procesamiento Proteico-Postraduccional , Proteínas Ubiquitinadas/fisiología , Carcinoma/etiología , Carcinoma/genética , Carcinoma/metabolismo , Carcinoma/terapia , Daño del ADN , Reparación del ADN , ADN de Neoplasias/genética , ADN de Neoplasias/metabolismo , Progresión de la Enfermedad , Humanos , Proteínas de Neoplasias/genética , Neoplasias/etiología , Neoplasias/genética , Neoplasias/terapia , Medicina de Precisión , Elongación de la Transcripción Genética , Ubiquitina Tiolesterasa/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , Ubiquitinación
13.
Methods Mol Biol ; 2265: 247-263, 2021.
Artículo en Inglés | MEDLINE | ID: mdl-33704720

RESUMEN

In recent years, circulating tumor DNA (ctDNA) has emerged as a promising prognostic and monitoring biomarker of various cancers, including melanoma. However, sensitive methods are required for its preservation, isolation, and detection. Here we describe a sensitive method for plasma ctDNA isolation using a column-based extraction kit, followed by quantification using a single mutational target with a droplet digital PCR system. This sensitive protocol has been successfully used to quantify diverse mutations present in plasma-derived ctDNA from cancer patients. The full procedure, from blood processing to the analysis of results, takes approximately a day of work.


Asunto(s)
ADN Tumoral Circulante/sangre , ADN de Neoplasias/sangre , Melanoma/sangre , Reacción en Cadena de la Polimerasa/métodos , Biomarcadores de Tumor/sangre , Biomarcadores de Tumor/genética , ADN de Neoplasias/genética , ADN de Neoplasias/aislamiento & purificación , Humanos , Melanoma/genética , Melanoma/patología , Plasma/metabolismo , Proteínas Proto-Oncogénicas B-raf/genética
14.
Radiol Med ; 126(6): 786-794, 2021 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-33512651

RESUMEN

PURPOSE: To develop a CT texture-based model able to predict epidermal growth factor receptor (EGFR)-mutated, anaplastic lymphoma kinase (ALK)-rearranged lung adenocarcinomas and distinguish them from wild-type tumors on pre-treatment CT scans. MATERIALS AND METHODS: Texture analysis was performed using proprietary software TexRAD (TexRAD Ltd, Cambridge, UK) on pre-treatment contrast-enhanced CT scans of 84 patients with metastatic primary lung adenocarcinoma. Textural features were quantified using the filtration-histogram approach with different spatial scale filters on a single 5-mm-thick central slice considered representative of the whole tumor. In order to deal with class imbalance regarding mutational status percentages in our population, the dataset was optimized using the synthetic minority over-sampling technique (SMOTE) and correlations with textural features were investigated using a generalized boosted regression model (GBM) with a nested cross-validation approach (performance averaged over 1000 resampling episodes). RESULTS: ALK rearrangements, EGFR mutations and wild-type tumors were observed in 19, 28 and 37 patients, respectively, in the original dataset. The balanced dataset was composed of 171 observations. Among the 29 original texture variables, 17 were employed for model building. Skewness on unfiltered images and on fine texture was the most important features. EGFR-mutated tumors showed the highest skewness while ALK-rearranged tumors had the lowest values with wild-type tumors showing intermediate values. The average accuracy of the model calculated on the independent nested validation set was 81.76% (95% CI 81.45-82.06). CONCLUSION: Texture analysis, in particular skewness values, could be promising for noninvasive characterization of lung adenocarcinoma with respect to EGFR and ALK mutations.


Asunto(s)
Quinasa de Linfoma Anaplásico/genética , Carcinoma de Pulmón de Células no Pequeñas/diagnóstico , ADN de Neoplasias/genética , Neoplasias Pulmonares/diagnóstico , Mutación , Tomografía Computarizada por Rayos X/métodos , Adulto , Anciano , Anciano de 80 o más Años , Quinasa de Linfoma Anaplásico/metabolismo , Carcinoma de Pulmón de Células no Pequeñas/genética , Carcinoma de Pulmón de Células no Pequeñas/metabolismo , Análisis Mutacional de ADN , Receptores ErbB/genética , Receptores ErbB/metabolismo , Femenino , Humanos , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/metabolismo , Masculino , Persona de Mediana Edad , Valor Predictivo de las Pruebas , Estudios Retrospectivos
15.
Proc Natl Acad Sci U S A ; 118(3)2021 01 19.
Artículo en Inglés | MEDLINE | ID: mdl-33408251

RESUMEN

Cisplatin is a mainstay of systemic therapy for a variety of cancers, such as lung cancer, head and neck cancer, and ovarian cancer. However, resistance to cisplatin represents one of the most significant barriers for patient outcome improvement. Actin-like 6A (ACTL6A) is a component of several chromatin remodeling complexes, including SWI/SNF, NuA4/TIP60 histone acetylase, and INO80. Amplification of ACTL6A gene is often seen in lung squamous cell carcinoma, ovarian cancer, and esophageal cancer, but its significance remains to be fully determined. Here we identify ACTL6A overexpression as a novel cause for platinum resistance. High levels of ACTL6A are associated with chemoresistance in several types of human cancer. We show that overexpression of ACTL6A leads to increased repair of cisplatin-DNA adducts and resistance to cisplatin treatment. In contrast, depletion of ACTL6A inhibits the repair of cisplatin-induced DNA lesions, and increases cisplatin sensitivity in cisplatin-resistant ovarian cancer cells. The regulation of repair by ACTL6A is mediated through the SWI/SNF chromatin remodeling complex. Treatment with a histone deacetylase inhibitor can reverse the effect of ACTL6A overexpression on the repair of cisplatin-induced DNA damage and render cancer cells more sensitive to cisplatin treatment in a xenograft mouse model. Taken together, our study uncovers a novel role for ACTL6A in platinum resistance, and provides evidence supporting the feasibility of using HDAC inhibitors for platinum resistant tumors.


Asunto(s)
Actinas/genética , Adenocarcinoma del Pulmón/genética , Carcinoma de Células Escamosas/genética , Proteínas Cromosómicas no Histona/genética , Reparación del ADN/efectos de los fármacos , Proteínas de Unión al ADN/genética , Resistencia a Antineoplásicos/genética , Neoplasias Pulmonares/genética , Neoplasias Ováricas/genética , ATPasas Asociadas con Actividades Celulares Diversas/genética , ATPasas Asociadas con Actividades Celulares Diversas/metabolismo , Actinas/metabolismo , Adenocarcinoma del Pulmón/tratamiento farmacológico , Adenocarcinoma del Pulmón/metabolismo , Adenocarcinoma del Pulmón/mortalidad , Animales , Antineoplásicos/uso terapéutico , Carcinoma de Células Escamosas/tratamiento farmacológico , Carcinoma de Células Escamosas/metabolismo , Carcinoma de Células Escamosas/mortalidad , Cromatina/química , Cromatina/efectos de los fármacos , Cromatina/metabolismo , Proteínas Cromosómicas no Histona/metabolismo , Cisplatino/uso terapéutico , Aductos de ADN , Daño del ADN , ADN de Neoplasias/genética , ADN de Neoplasias/metabolismo , Proteínas de Unión al ADN/metabolismo , Resistencia a Antineoplásicos/efectos de los fármacos , Femenino , Regulación Neoplásica de la Expresión Génica , Inhibidores de Histona Desacetilasas/uso terapéutico , Humanos , Neoplasias Pulmonares/tratamiento farmacológico , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/mortalidad , Lisina Acetiltransferasa 5/genética , Lisina Acetiltransferasa 5/metabolismo , Ratones , Neoplasias Ováricas/tratamiento farmacológico , Neoplasias Ováricas/metabolismo , Neoplasias Ováricas/mortalidad , Panobinostat/uso terapéutico , Análisis de Supervivencia , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Ensayos Antitumor por Modelo de Xenoinjerto
16.
PLoS One ; 16(1): e0245667, 2021.
Artículo en Inglés | MEDLINE | ID: mdl-33481917

RESUMEN

BACKGROUND: Large inter-individual variations in drug metabolism pose a challenge in determining 6-mercaptopurine (6MP) doses. As the last product of 6MP metabolism, DNA-thioguanine nucleotide (DNA-TGN) could reflect the efficacy of 6MP, especially in patients harboring variants in the 6MP metabolism pathway. The aim of this study was to investigate the clinical significance of DNA-TGN monitoring in Korean pediatric acute lymphoblastic leukemia (ALL) patients, focusing on the NUDT15 genotype. METHODS: The subjects of this study were patients who underwent ALL treatment with 6MP. Tests for the NUDT15 and TPMT genotypes were performed, and prospective DNA-TGN and erythrocyte TGN samples were collected after two weeks or more of 6MP treatment. DNA-TGN was quantified using the liquid chromatography-tandem mass spectrometry method. RESULTS: A total of 471 DNA-TGN measurements in 71 patients were analyzed, which ranged from 1.0 to 903.1 fmol thioguanine/µg DNA. The 6MP intensity demonstrated a significant relationship with DNA-TGN concentration (P<0.001). Patients harboring NUDT15 variants were treated with a lower dose of 6MP (P<0.001); however, there was no significant difference in DNA-TGN concentration when compared to patients carrying wild-type NUDT15 (P = 0.261). These patients also presented higher variation in DNA-TGN levels (P = 0.002) and DNA-TGN/6MP intensity (P = 0.019) compared to patients carrying wild-type NUDT15. DNA-TGN concentration did not show a significant correlation with WBC count (P = 0.093). CONCLUSIONS: Patients harboring NUDT15 variants demonstrated similar DNA-TGN concentrations even at low doses of 6MP and showed high variability in DNA-TGN. Particularly in patients with NUDT15 variants who need a reduced 6MP dose, DNA-TGN could be applied as a useful marker to monitor the therapeutic effect of 6MP.


Asunto(s)
Biomarcadores de Tumor , ADN de Neoplasias , Genotipo , Mercaptopurina/administración & dosificación , Proteínas de Neoplasias , Leucemia-Linfoma Linfoblástico de Células Precursoras , Pirofosfatasas , Tioguanina/metabolismo , Adolescente , Adulto , Biomarcadores de Tumor/genética , Biomarcadores de Tumor/metabolismo , Niño , Preescolar , ADN de Neoplasias/genética , ADN de Neoplasias/metabolismo , Femenino , Humanos , Lactante , Masculino , Metiltransferasas/genética , Metiltransferasas/metabolismo , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Leucemia-Linfoma Linfoblástico de Células Precursoras/tratamiento farmacológico , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras/metabolismo , Pirofosfatasas/genética , Pirofosfatasas/metabolismo
17.
Exp Eye Res ; 203: 108426, 2021 02.
Artículo en Inglés | MEDLINE | ID: mdl-33387485

RESUMEN

PURPOSE: Uveal melanoma (UM) is an aggressive malignancy, in which nearly 50% of the patients die from metastatic disease. Aberrant DNA methylation is recognized as an important epigenomic event in carcinogenesis. Formalin-fixed paraffin-embedded (FFPE) samples represent a valuable source of tumor tissue, and recent technology has enabled the use of these samples in genome-wide DNA methylation analyses. Our aim was to investigate differential DNA methylation in relation to histopathological classification and survival data. In addition we sought to identify aberrant DNA methylation of genes that could be associated with metastatic disease and poor survival. METHODS: FFPE samples from UM patients (n = 23) who underwent enucleation of the eye in the period 1976-1989 were included. DNA methylation was assessed using the Illumina Infinium HumanMethylation450 array and coupled to histopathological data, Cancer Registry of Norway- (registered UM metastasis) and Norwegian Cause of Death Registry- (time and cause of death) data. Differential DNA methylation patterns contrasting histological classification, survival data and clustering properties were investigated. Survival groups were defined as "Early metastasis" (metastases and death within 2-5 years after enucleation, n = 8), "Late metastasis" (metastases and death within 9-21 years after enucleation, n = 7) and "No metastasis" (no detected metastases ≥18 years after enucleation, n = 8). A subset of samples were selected based on preliminary multi-dimensional scaling (MDS) plots, histopathological classification, chromosome 3 status, survival status and clustering properties; "Subset Early metastasis" (n = 4) vs "Subset No metastasis" (n = 4). Bioinformatics analyses were conducted in the R statistical software. Differentially methylated positions (DMPs) and differentially methylated regions (DMRs) in various comparisons were assessed. Gene expression of relevant subgroups was determined by microarray analysis and quantitative reverse-transcription polymerase chain reaction (qRT-PCR). RESULTS: DNA methylation analyses identified 2 clusters that separated the samples according to chromosome 3 status. Cluster 1 consisted of samples (n = 5) with chromosome 3 disomy (D3), while Cluster 2 was comprised of samples (n = 15) with chromosome 3 monosomy (M3). 1212 DMRs and 9386 DMPs were identified in M3 vs D3. No clear clusters were formed based on our predefined survival groups ("Early", "Late", "No") nor histopathological classification (Epithelioid, Mixed, Spindle). We identified significant changes in DNA methylation (beta FC ≥ 0.2, adjusted p < 0.05) between two sample subsets (n = 8). "Subset Early metastasis" (n = 4) vs "Subset No metastasis" (n = 4) identified 348 DMPs and 36 DMRs, and their differential gene expression by microarray showed that 14 DMPs and 2 DMRs corresponded to changes in gene expression (FC ≥ 1.5, p < 0.05). RNF13, ZNF217 and HYAL1 were hypermethylated and downregulated in "Subset Early metastasis" vs "Subset No metastasis" and could be potential tumor suppressors. TMEM200C, RGS10, ADAM12 and PAM were hypomethylated and upregulated in "Subset Early metastasis vs "Subset No metastasis" and could be potential oncogenes and thus markers of early metastasis and poor prognosis in UM. CONCLUSIONS: DNA methylation profiling showed differential clustering of samples according to chromosome 3 status: Cluster 1 (D3) and Cluster 2 (M3). Integrated differential DNA methylation and gene expression of two subsets of samples identified genes associated with early metastasis and poor prognosis. RNF13, ZNF217 and HYAL1 are hypermethylated and candidate tumor suppressors, while TMEM200C, RGS10, ADAM12 and PAM are hypomethylated and candidate oncogenes linked to early metastasis. UM FFPE samples represent a valuable source for methylome studies and enable long-time follow-up.


Asunto(s)
Metilación de ADN , ADN de Neoplasias/genética , Regulación Neoplásica de la Expresión Génica/fisiología , Melanoma/genética , Proteínas de Neoplasias/genética , Neoplasias de la Úvea/genética , Adulto , Variaciones en el Número de Copia de ADN , Epigenómica , Enucleación del Ojo , Femenino , Formaldehído , Perfilación de la Expresión Génica , Humanos , Masculino , Melanoma/patología , Melanoma/cirugía , Persona de Mediana Edad , Análisis de Secuencia por Matrices de Oligonucleótidos , Adhesión en Parafina , Análisis de Componente Principal , Reacción en Cadena en Tiempo Real de la Polimerasa , Fijación del Tejido , Neoplasias de la Úvea/patología , Neoplasias de la Úvea/cirugía , Adulto Joven
18.
Mol Biol Rep ; 48(1): 709-720, 2021 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-33389482

RESUMEN

I. BACKGROUND: A combination of etoposide (VP-16) and cisplatin (CDDP) is the standard treatment for certain colon cancers. These drugs promote the death of cancer cells via direct and indirect induction of the most lethal DNA lesions - DNA double-stand breaks. However, cancer cells can reverse the DNA damaging effect of anticancer drugs by triggering DNA repair processes. In eukaryotic cells, the main DNA repair pathway responsible for DNA double-stand breaks repair is non-homologous end-joining (NHEJ). Inhibitors of DNA repair are of special interest in cancer research as they could break the cellular resistance to DNA-damaging agents and increase the efficiency of standard cancer treatments. In this study, we investigated the effect of two NHEJ inhibitors, SCR7 and NU7441, on the cytotoxic mechanism of VP-16/CDDP in a LoVo human colorectal adenocarcinoma cell line. SCR7 blocks Ligase IV-mediated joining by interfering with its DNA binding, whereas NU7441 is a highly potent and selective DNA-PK inhibitor.II. METHODS AND RESULTS: Both inhibitors synergistically increased the cytotoxicity of CDDP and VP-16 when combined, but the effect of SCR7 was more pronounced. SCR7 and NU7441 also significantly increased VP-16; CDDP induced DNA double-stand breaks level and delayed drug-induced DSB repair, as seen on the comet assay and measured using H2AX foci. We also observed changes in cell cycle distribution and enhanced apoptosis ratio in colorectal adenocarcinoma cells treated with DNA repair inhibitors and VP-16/CDDP.III. CONCLUSIONS: Our data support the hypothesis that NHEJ inhibitors could be used in conjunction with standard therapy to provide effective clinical improvement and allow reduction in drug doses.


Asunto(s)
Antineoplásicos/farmacología , Cromonas/farmacología , Cisplatino/farmacología , Reparación del ADN por Unión de Extremidades/efectos de los fármacos , ADN de Neoplasias/genética , Etopósido/farmacología , Morfolinas/farmacología , Pirimidinas/farmacología , Bases de Schiff/farmacología , Protocolos de Quimioterapia Combinada Antineoplásica , Línea Celular Tumoral , Ensayo Cometa , Roturas del ADN de Doble Cadena , Fragmentación del ADN/efectos de los fármacos , ADN de Neoplasias/metabolismo , Sinergismo Farmacológico , Células Epiteliales/efectos de los fármacos , Células Epiteliales/metabolismo , Células Epiteliales/patología , Histonas/genética , Histonas/metabolismo , Humanos
19.
Nat Methods ; 18(2): 144-155, 2021 02.
Artículo en Inglés | MEDLINE | ID: mdl-33398189

RESUMEN

Subclonal reconstruction from bulk tumor DNA sequencing has become a pillar of cancer evolution studies, providing insight into the clonality and relative ordering of mutations and mutational processes. We provide an outline of the complex computational approaches used for subclonal reconstruction from single and multiple tumor samples. We identify the underlying assumptions and uncertainties in each step and suggest best practices for analysis and quality assessment. This guide provides a pragmatic resource for the growing user community of subclonal reconstruction methods.


Asunto(s)
ADN de Neoplasias/genética , Neoplasias/genética , Análisis de Secuencia de ADN/métodos , Algoritmos , Humanos , Polimorfismo de Nucleótido Simple
20.
Pediatr Blood Cancer ; 68(1): e28741, 2021 01.
Artículo en Inglés | MEDLINE | ID: mdl-33009870

RESUMEN

BACKGROUND: Pediatric papillary thyroid carcinoma (PTC) is clinically and biologically distinct from adult PTC. We sequenced a cohort of clinically annotated pediatric PTC cases enriched for high-risk tumors to identify genetic alterations of relevance for diagnosis and therapy. METHODS: Tumor DNA and RNA were extracted from FFPE tissue and subjected to next-generation sequencing (NGS) library preparation using a custom 124-gene hybridization capture panel and the 75-gene Archer Oncology Research Panel, respectively. NGS libraries were sequenced on an Illumina MiSeq. RESULTS: Thirty-six pediatric PTC cases were analyzed. Metastases were frequently observed to cervical lymph nodes (29/36, 81%), with pulmonary metastases less commonly found (10/36, 28%). Relapsed or refractory disease occurred in 18 patients (18/36, 50%). DNA sequencing revealed targetable mutations in 8 of 31 tumors tested (26%), most commonly BRAF p.V600E (n = 6). RNA sequencing identified targetable fusions in 13 of 25 tumors tested (52%): RET (n = 8), NTRK3 (n = 4), and BRAF. Mutually exclusive targetable alterations were discovered in 15 of the 20 tumors (75%) with both DNA and RNA analyzed. Fusion-positive PTC was associated with multifocal disease, higher tumor staging, and higher American Thyroid Association risk levels. Both BRAF V600E mutations and gene fusions were correlated with the presence of cervical metastases. CONCLUSIONS: Targetable alterations were identified in 75% of pediatric PTC cases with both DNA and RNA evaluated. Inclusion of RNA sequencing for detection of fusion genes is critical for evaluation of these tumors. Patients with fusion-positive tumors were more likely to have features of high-risk disease.


Asunto(s)
Biomarcadores de Tumor/genética , Carcinoma Papilar/patología , ADN de Neoplasias/análisis , Neoplasias Pulmonares/secundario , Mutación , Análisis de Secuencia de ARN/métodos , Neoplasias de la Tiroides/patología , Adolescente , Adulto , Carcinoma Papilar/genética , Niño , Preescolar , ADN de Neoplasias/genética , Femenino , Estudios de Seguimiento , Humanos , Lactante , Recién Nacido , Neoplasias Pulmonares/genética , Metástasis Linfática , Masculino , Pronóstico , Estudios Retrospectivos , Neoplasias de la Tiroides/genética , Adulto Joven
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