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Medullary Thyroid Carcinoma (MTC) is a rare neuroendocrine tumour whose diagnosis includes evaluating calcitonin serum levels, which can present fluctuations unrelated to MTC. Here, we investigated circulating DNA fragmentation and methylation changes as potential biomarkers using ddPCR on cell-free DNA (cfDNA) isolated from the plasma of MTC patients. For cfDNA fragmentation analysis, we investigated the fragment size distribution of a gene family and calculated short fragment fraction (SFF). Methylation analyses evaluated the methylation levels of CG_16698623, a CG dinucleotide in the MGMT gene that we found hypermethylated in MTC tissues by analyzing public databases. The SFF ratio and methylation of CG_16698623 were significantly increased in plasma from MTC patients at diagnosis, and patients with clinical remission or stable disease at follow-up showed no significant SFF difference compared with healthy subjects. Our data support the diagnostic value of cfDNA traits that could enable better management of MTC patients.
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Medullary thyroid carcinoma (MTC) is a malignant tumor with challenging management. Multi-targeted kinase inhibitors (MKI) and tyrosine-kinase inhibitors (TKI) with high specificity for RET protein are approved for advanced MTC treatment. However, their efficacy is hindered by evasion mechanisms of tumor cells. Thus, the aim of this study was the identification of an escape mechanism in MTC cells exposed to a highly selective RET TKI. TT cells were treated with TKI, MKI, and/or the HH-Gli inhibitors, GANT61 and Arsenic Trioxide (ATO), in the presence or absence of hypoxia. RET modifications, oncogenic signaling activation, proliferation and apoptosis were assessed. Additionally, cell modifications and HH-Gli activation were also evaluated in pralsetinib-resistant TT cells. Pralsetinib inhibited RET autophosphorylation and RET downstream pathways activation in normoxic and hypoxic conditions. Additionally, pralsetinib impaired proliferation, induced the activation of apoptosis and, in hypoxic cells, downregulated HIF-1α. Focusing on escape molecular mechanisms associated with therapy, we observed increased Gli1 levels in a subset of cells. Indeed, pralsetinib stimulated the re-localization of Gli1 into the cell nuclei. Treatment of TT cells with both pralsetinib and ATO resulted in Gli1 down-regulation and impaired cell viability. Moreover, pralsetinib-resistant cells confirmed Gli1 activation and up-regulation of its transcriptionally regulated target genes. Altogether, we showed that pralsetinib impairs MTC cell growth and induces cell death, also in hypoxic conditions. The HH-Gli pathway is a new molecular mechanism of escape to pralsetinib therapy that can be overcome through combined therapy.
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Neoplasias de la Tiroides , Humanos , Proteína con Dedos de Zinc GLI1/metabolismo , Transducción de Señal , Trióxido de Arsénico , Neoplasias de la Tiroides/genéticaRESUMEN
PURPOSE: In this study, we evaluated the biological role of miRNA-31-5p in papillary thyroid cancer (PTC). METHODS: By using the real-time PCR, we measured miRNA-31-5p expression levels in 25 PTC tissues and in two human PTC cell lines (K1 and TPC-1). Then, K1 cells were transiently transfected with mirVana inhibitor or mirVana mimic to miRNA-31-5-p. Cell proliferation was determined by MTT and colony formation assays. The in vitro metastatic ability of thyroid cancer cells was evaluated by adhesion, migration and invasion assays. Epithelial mesenchymal transition (EMT) and Hippo pathway related gene and protein levels were evaluated by using the TaqMan™ Gene Expression Assays and western blot analysis, respectively. RESULTS: We found a significant increase of miR-31-5-p expression in tumor tissue and in K1 cells harboring the BRAF p.V600E mutation. Knockdown of miR-31-5p determined a reduction of cell proliferation, associated with a significant decrease in cell adhesion, migration and invasion properties. A downregulation of EMT markers and YAP/ß-catenin axis was also observed. CONCLUSIONS: Our findings suggest that miRNA-31-5p acts as oncogenic miRNA in human thyrocytes and its overexpression may be involved in the BRAF-related tumorigenesis in PTCs, providing new understanding into its pathological role in PTC progression and invasiveness.
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MicroARNs , Neoplasias de la Tiroides , Humanos , Cáncer Papilar Tiroideo/genética , Cáncer Papilar Tiroideo/patología , Proteínas Proto-Oncogénicas B-raf/genética , Línea Celular Tumoral , Movimiento Celular/genética , Neoplasias de la Tiroides/patología , MicroARNs/genética , MicroARNs/metabolismo , Proliferación Celular/genética , Invasividad Neoplásica/genética , Regulación Neoplásica de la Expresión GénicaRESUMEN
Aberrant activation of the RET proto-oncogene is implicated in a plethora of cancers. RET gain-of-function point mutations are driver events in multiple endocrine neoplasia 2 (MEN2) syndrome and in sporadic medullary thyroid cancer, while RET rearrangements are driver events in several non-medullary thyroid cancers. Drugs able to inhibit RET have been used to treat RET-mutated cancers. Multikinase inhibitors were initially used, though they showed modest efficacy and significant toxicity. However, new RET selective inhibitors, such as selpercatinib and pralsetinib, have recently been tested and have shown good efficacy and tolerability, even if no direct comparison is yet available between multikinase and selective inhibitors. The advent of high-throughput technology has identified cancers with rare RET alterations beyond point mutations and fusions, including RET deletions, raising questions about whether these alterations have a functional effect and can be targeted by RET inhibitors. In this mini review, we focus on tumors with RET deletions, including deletions/insertions (indels), and their response to RET inhibitors.
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Here, we describe a protocol to generate organoids from human thyroid cancer cells. Starting from the same patient-derived cells, we establish both organoids and primary lines. The organoid medium is supplemented with conditioned medium obtained from the primary cell line. This modification enables culture of the organoid lines for up to 10 months. Even after long-term culture, the organoids retain the genetic and phenotypic characteristics of their tissue of origin.
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Organoides , Neoplasias de la Tiroides , Medios de Cultivo Condicionados/metabolismo , Humanos , Neoplasias de la Tiroides/genéticaRESUMEN
The release of molecules in exosomal cargoes is involved in tumor development and progression. We compared the profiles of exosomal microRNAs released by two thyroid cancer cell lines (TPC-1 and K1) with that of non-tumorigenic thyroid cells (Nthy-ori-3-1), and we explored the network of miRNA-target interaction. After extraction and characterization of exosomes, expression levels of microRNAs were investigated using custom TaqMan Advanced array cards, and compared with those expressed in the total cell extracts. The functional enrichment and network-based analysis of the miRNAs' targets was also performed. Five microRNAs (miR-21-5p, miR-31-5p, miR-221-3p, miR-222-3p, and let-7i-3p) were significantly deregulated in the exosomes of tumor cells vs. non-tumorigenic cells, and three of them (miR-31-5p, miR-222-3p, and let-7i-3p) in the more aggressive K1 compared to TPC-1 cells. The network analysis of the five miRNAs identified some genes as targets of more than one miRNAs. These findings permitted the identification of exosomal microRNAs secreted by aggressive PTC cells, and indicated that their main targets are regulators of the tumor microenvironment. A deeper analysis of the functional role of the targets of exosomal miRNAs will provide further information on novel targets of molecular treatments for these neoplasms.
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PURPOSE: In this study, we investigated the profile of microRNAs (miRNAs) contained in exosomes secreted in the serum of patients with papillary thyroid cancer (PTC). METHODS: Exosome were isolated by adding ExoQuick Exosome Precipitation Solution. Dynamic light scattering (DLS) and western blotting analysis were used to ensure the quality of exosomes. The expression levels of miRNAs were investigated using custom-designed TaqMan Advanced miRNA Array Cards in the screening cohort and using specific TaqMan Advanced MicroRNA Assays in the validation cohort. RESULTS: We identified miR24-3p, miR146a-5p, miR181a-5p and miR382-5p with different expression levels in two different series of 56 and 58 PTC patients as compared with healthy controls. Significant differences in the expression of three PTC exosomal miRNAs, depending on the presence of lymph node metastasis, were detected in only one PTC series. When comparing the expression levels of some PTC-specific exosomal miRNAs with those of the same miRNAs circulating free of any encapsulation, we found a significant correlation for only miR24-3p, suggesting that only select miRNAs are secreted in exosomes. CONCLUSIONS: Our findings demonstrate that four miRNAs are differently secreted in the exosomes of PTC patients, whereas no conclusive results were found to characterize PTCs with lymph node metastasis, suggesting caution in the use of circulating exosomal miRNA expression levels as lymph node metastasis biomarkers. Further investigation into the mechanisms governing miRNA secretion in tumor cells are required.
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MicroARN Circulante , Exosomas , MicroARNs , Neoplasias de la Tiroides , Exosomas/metabolismo , Humanos , MicroARNs/genética , Cáncer Papilar Tiroideo/metabolismo , Neoplasias de la Tiroides/patologíaRESUMEN
The SARS-CoV-2 pandemic is a worldwide public health emergency. Despite the beginning of a vaccination campaign, the search for new drugs to appropriately treat COVID-19 patients remains a priority. Drug repurposing represents a faster and cheaper method than de novo drug discovery. In this study, we examined three different network-based approaches to identify potentially repurposable drugs to treat COVID-19. We analyzed transcriptomic data from whole blood cells of patients with COVID-19 and 21 other related conditions, as compared with those of healthy subjects. In addition to conventionally used drugs (e.g., anticoagulants, antihistaminics, anti-TNFα antibodies, corticosteroids), unconventional candidate compounds, such as SCN5A inhibitors and drugs active in the central nervous system, were identified. Clinical judgment and validation through clinical trials are always mandatory before use of the identified drugs in a clinical setting.
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Tratamiento Farmacológico de COVID-19 , COVID-19 , Simulación por Computador , Reposicionamiento de Medicamentos , Antiinflamatorios/farmacología , COVID-19/prevención & control , Fármacos del Sistema Nervioso Central/farmacología , Reposicionamiento de Medicamentos/métodos , Reposicionamiento de Medicamentos/tendencias , Inhibidores Enzimáticos/farmacología , Perfilación de la Expresión Génica/métodos , Humanos , Factores Inmunológicos/farmacología , Resultado del Tratamiento , Bloqueadores del Canal de Sodio Activado por Voltaje/farmacologíaRESUMEN
Cancer stem-like cells (CSCs) have self-renewal abilities responsible for cancer progression, therapy resistance, and metastatic growth. The glioblastoma stem-like cells are the most studied among CSC populations. A recent study identified four transcription factors (SOX2, SALL2, OLIG2, and POU3F2) as the minimal core sufficient to reprogram differentiated glioblastoma (GBM) cells into stem-like cells. Transcriptomic data of GBM tissues and cell lines from two different datasets were then analyzed by the SWItch Miner (SWIM), a network-based software, and FOSL1 was identified as a putative regulator of the previously identified minimal core. Herein, we selected NTERA-2 and HEK293T cells to perform an in vitro study to investigate the role of FOSL1 in the reprogramming mechanisms. We transfected the two cell lines with a constitutive FOSL1 cDNA plasmid. We demonstrated that FOSL1 directly regulates the four transcription factors binding their promoter regions, is involved in the deregulation of several stemness markers, and reduces the cells' ability to generate aggregates increasing the extracellular matrix component FN1. Although further experiments are necessary, our data suggest that FOSL1 reprograms the stemness by regulating the core of the four transcription factors.
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Reprogramación Celular/genética , Células Madre Neoplásicas/fisiología , Proteínas Proto-Oncogénicas c-fos/fisiología , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/patología , Diferenciación Celular/genética , Línea Celular Tumoral , Regulación Neoplásica de la Expresión Génica , Glioblastoma/genética , Glioblastoma/patología , Células HEK293 , Células HeLa , Humanos , Células Madre Neoplásicas/patología , Proteínas Proto-Oncogénicas c-fos/genéticaRESUMEN
The Cancer Genome Atlas database offers the possibility of analyzing genome-wide expression RNA-Seq cancer data using paired counts, that is, studies where expression data are collected in pairs of normal and cancer cells, by taking samples from the same individual. Correlation of gene expression profiles is the most common analysis to study co-expression groups, which is used to find biological interpretation of -omics big data. The aim of the paper is threefold: firstly we show for the first time, the presence of a "regulation-correlation bias" in RNA-Seq paired expression data, that is an artifactual link between the expression status (up- or down-regulation) of a gene pair and the sign of the corresponding correlation coefficient. Secondly, we provide a statistical model able to theoretically explain the reasons for the presence of such a bias. Thirdly, we present a bias-removal algorithm, called SEaCorAl, able to effectively reduce bias effects and improve the biological significance of correlation analysis. Validation of the SEaCorAl algorithm is performed by showing a significant increase in the ability to detect biologically meaningful associations of positive correlations and a significant increase of the modularity of the resulting unbiased correlation network.
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Perfilación de la Expresión Génica , Genoma , Algoritmos , Humanos , RNA-Seq , Análisis de Secuencia de ARN , TranscriptomaRESUMEN
BACKGROUND: Understanding the molecular mechanisms underlying papillary thyroid cancer (PTC) proved to be vital not only for diagnostic purposes but also for tailored treatments. Despite the strong evidence of heritability, only a small subset of alterations has been implicated in PTC pathogenesis. To this reason, we used targeted next-generation sequencing (NGS) to identify candidate variants implicated in PTC pathogenesis, progression, and invasiveness. METHODS: A total of 42 primary PTC tissues were investigated using a targeted next-generation sequencing (NGS) panel enlisting 47 genes involved in DNA repair and tumor progression. RESULTS: We identified 57 point mutations in 78.5% of samples (n = 32). Thirty-two somatic mutations were identified exclusively in known thyroid cancer genes (BRAF, KRAS, NRAS, and TERT). Unpredictably, 45% of the all identified mutations (n = 25) resulted to be germline, most affecting DNA repair genes. Interestingly, none of the latter variants was in the main population databases. Following ACMG classification, 20% of pathogenic/likely pathogenic and 68% of variant of unknown significance were identified. CONCLUSIONS: Overall, our results support the hypothesis that rare germline variants in DNA repair genes are accountable for PTC susceptibility. More data, including the segregation analysis in affected families, should be collected before definitely annotate these alterations and to establish their potential prognostic and treatment implications.
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Neoplasias de la Tiroides , Reparación del ADN/genética , Células Germinativas , Mutación de Línea Germinal , Humanos , Mutación , Proteínas Proto-Oncogénicas B-raf/genética , Cáncer Papilar Tiroideo/genética , Neoplasias de la Tiroides/genéticaRESUMEN
Despite impressive efforts invested in epigenetic research in the last 50 years, clinical applications are still lacking. Only a few university hospital centers currently use epigenetic biomarkers at the bedside. Moreover, the overall concept of precision medicine is not widely recognized in routine medical practice and the reductionist approach remains predominant in treating patients affected by major diseases such as cancer and cardiovascular diseases. By its' very nature, epigenetics is integrative of genetic networks. The study of epigenetic biomarkers has led to the identification of numerous drugs with an increasingly significant role in clinical therapy especially of cancer patients. Here, we provide an overview of clinical epigenetics within the context of network analysis. We illustrate achievements to date and discuss how we can move from traditional medicine into the era of network medicine (NM), where pathway-informed molecular diagnostics will allow treatment selection following the paradigm of precision medicine.
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Biomarcadores , Enfermedades Cardiovasculares/genética , Enfermedades Cardiovasculares/terapia , Neoplasias/genética , Neoplasias/terapia , Sistemas de Atención de Punto , Medicina de Precisión/métodos , Epigénesis Genética , HumanosRESUMEN
Hürthle cell carcinomas (HCC) are rare differentiated thyroid cancers that display low avidity for radioactive iodine and respond poorly to kinase inhibitors. Here, using next-generation sequencing, we analyzed the mutational status of primary tissue and poorly differentiated metastatic tissue from two HCC patients. In both cases, metastatic tissues harbored a mutation of SETD2, each resulting in loss of the SRI and WW domains of SETD2, a methyltransferase that trimethylates H3K36 (H3K36me3) and also interacts with p53 to promote its stability. Functional studies of the novel p.D1890fs6* mutation (case 1) revealed significantly reduced H3K36me3 levels in SETD2-mutated tissue and primary cell cultures and decreased levels of the active form of p53. Restoration of SETD2-wildtype expression in the SETD2-mutant cells significantly reduced the expression of four well-known stemness markers (OCT-4, SOX2, IPF1, Goosecoid). These findings suggest potential roles for SETD2 loss-of-function mutations in HCC progression, possibly involving p53 destabilization and promotion of stemness. Their prevalence and potential treatment implications in thyroid cancer, especially HCC, require further study.
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PURPOSE: The identification of somatic mutations in cancer specimens enables detection of molecular markers for personalized treatment. We recently developed a novel molecular assay and evaluated its clinical performance as an ancillary molecular method for indeterminate thyroid nodule cytology. Herein we describe the analytical validation of the novel targeted next-generation sequencing (NGS) assay in thyroid samples from different sources. METHODS: We present validation data of a novel NGS-based panel on 463 thyroid samples, including 310 fine-needle aspiration (FNA) specimens from different sources (FNA collected in preservative solution, liquid-based, and stained smears), 10 fresh frozen, and 143 formalin-fixed paraffin-embedded (FFPE) thyroid tissue specimens. Sequencing performance in the different samples was evaluated along with reproducibility, repeatability, minimum nucleic acid input to detect variants, and analytical sensitivity of the assay. RESULTS: All thyroid samples achieved high sequencing performance, with a mean base coverage depth ranging from 2228 × (in liquid-based FNA) to 3661 × (in FNA stained smears), and coverage uniformity ranging from 86% (in FFPE) to 95% (in FNA collected in preservative solution), with all target regions covered above the minimum depth required to call a variant (500×). The minimum nucleic acid input was 1 ng. Analytic sensitivity for mutation detection was 2-5% mutant allele frequency. CONCLUSIONS: This validation study of a novel NGS-based assay for thyroid nodules demonstrated that the assay can be reliably used on multiple thyroid sample types, including FNA from different sources and FF and FFPE thyroid samples, thus providing a robust and reliable assay to genotype thyroid nodules, which may improve thyroid cancer diagnosis and care.
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Nódulo Tiroideo , Biopsia con Aguja Fina , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Mutación , Reproducibilidad de los Resultados , Nódulo Tiroideo/diagnóstico , Nódulo Tiroideo/genéticaRESUMEN
PURPOSE: Deciding whether patients with a cytologically indeterminate thyroid nodule should be referred for surgery or for active surveillance is an important challenge for clinicians. The aim of this study was to evaluate the performance of a novel dual-component molecular assay as an ancillary molecular method for resolving indeterminate thyroid nodule cytology. METHODS: We selected 156 thyroid nodules from those that had undergone fine-needle aspiration processed by liquid-based cytology and surgical resection between June 2016 and December 2017. The sample set included 63 nodules cytologically classified as indeterminate, and 93 other nodules randomly selected from those with non-diagnostic, benign, suspicious, or malignant cytology. Nucleic acids from each nodule were subjected to next-generation sequencing analysis for mutation detection in 23 genes and to digital polymerase chain reaction (PCR) evaluation for miR-146b-5p expression levels. RESULTS: Used alone, mutation analysis in the indeterminate subset (cancer prevalence: 22.5%) displayed high sensitivity (89%) and NPV (96%). In contrast, the miR-146b-5p assay offered high specificity (93%) and PPV (93%). Combined use of both analyses improved panel performance by eliminating false-negative results. CONCLUSIONS: These preliminary data suggest that a dual-component molecular test can increase the diagnostic accuracy of thyroid cytology alone by reducing the number of nodules that will be classified as indeterminate and increasing those that can be reliably classified as benign. If these findings are confirmed, this test can be considered for use in clinical practice and is expected to reduce diagnostic surgery and health care costs, and to improve patient quality of life.
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Neoplasias de la Tiroides , Nódulo Tiroideo , Biopsia con Aguja Fina , Análisis Mutacional de ADN , Humanos , Calidad de Vida , Sensibilidad y Especificidad , Neoplasias de la Tiroides/diagnóstico , Neoplasias de la Tiroides/genética , Nódulo Tiroideo/diagnóstico , Nódulo Tiroideo/genéticaRESUMEN
The incidence of papillary thyroid microcarcinoma (microPTC) has dramatically increased in the last decades. Most of these tumors remain small and clinically "silent", only small number progress. Although thyroid surgery used to be the only therapeutic approach, recent guidelines now consider active surveillance for low-risk microPTC. For this reason, more accurate risk stratification of microPTC is needed. The optimal management of low-risk microPTC through accurate risk stratification represents a major clinical issue.
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Carcinoma Papilar/diagnóstico , Carcinoma Papilar/terapia , Neoplasias de la Tiroides/diagnóstico , Neoplasias de la Tiroides/terapia , Carcinoma Papilar/patología , Carcinoma Papilar/cirugía , Humanos , Estadificación de Neoplasias , Factores de Riesgo , Neoplasias de la Tiroides/patología , Neoplasias de la Tiroides/cirugía , TiroidectomíaRESUMEN
An amendment to this paper has been published and can be accessed via a link at the top of the paper.