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Platin-based chemotherapy is the standard treatment for patients with non-small cell lung cancer (NSCLC). However, resistance to this therapy is a major obstacle in successful treatment. In this study, we aimed to investigate the impact of several pharmacogenetic variants in patients with unresectable NSCLC treated with platin-based chemotherapy. Our results showed that DPYD variant carriers had significantly shorter progression-free survival and overall survival compared to DPYD wild-type patients, whereas DPD deficiency was not associated with a higher incidence of high-grade toxicity. For the first time, our study provides evidence that DPYD gene variants are associated with resistance to platin-based chemotherapy in NSCLC patients. Although further studies are needed to confirm these findings and explore the underlying mechanisms of this association, our results suggest that genetic testing of DPYD variants may be useful for identifying patients at a higher risk of platin-based chemotherapy resistance and might be helpful in guiding future personalized treatment strategies in NSCLC patients.
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Carcinoma Pulmonar de Células não Pequenas , Neoplasias Pulmonares , Humanos , Di-Hidrouracila Desidrogenase (NADP)/genética , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Carcinoma Pulmonar de Células não Pequenas/genética , Fluoruracila/uso terapêutico , Prognóstico , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/induzido quimicamente , Células GerminativasRESUMO
Despite advances in non-small cell lung cancer (NSCLC) research, this is still the most common cancer type that has been diagnosed up to date. microRNAs have emerged as useful clinical biomarkers in both tissue and liquid biopsy. However, there are no reliable predictive biomarkers for clinical use. We evaluated the preclinical use of seven candidate miRNAs previously identified by our group. We collected a total of 120 prospective samples from 88 NSCLC patients. miRNA levels were analyzed via qRT-PCR from tissue and blood samples. miR-124 gene target prediction was performed using RNA sequencing data from our group and interrogating data from 2952 NSCLC patients from two public databases. We found higher levels of all seven miRNAs in tissue compared to plasma samples, except for miR-124. Our findings indicate that levels of miR-124, both free-circulating and within exosomes, are increased throughout the progression of the disease, suggesting its potential as a marker of disease progression in both advanced and early stages. Our bioinformatics approach identified KPNA4 and SPOCK1 as potential miR-124 targets in NSCLC. miR-124 levels can be used to identify early-stage NSCLC patients at higher risk of relapse.
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Carcinoma Pulmonar de Células não Pequenas , Exossomos , Neoplasias Pulmonares , MicroRNAs , Humanos , Carcinoma Pulmonar de Células não Pequenas/diagnóstico , Carcinoma Pulmonar de Células não Pequenas/genética , Carcinoma Pulmonar de Células não Pequenas/patologia , Neoplasias Pulmonares/diagnóstico , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patologia , Prognóstico , Estudos Prospectivos , Biomarcadores Tumorais/metabolismo , Recidiva Local de Neoplasia/metabolismo , MicroRNAs/metabolismo , Exossomos/metabolismo , Biópsia Líquida , Proteoglicanas/metabolismo , alfa Carioferinas/metabolismoRESUMO
High myopia is the most severe and pathological form of myopia. It occurs when the spherical refractive error exceeds -6.00 spherical diopters (SDs) or the axial length (AL) of the eye is greater than 26 mm. This article focuses on early-onset high myopia, an increasingly common condition that affects children under 10 years of age and can lead to other serious ocular pathologies. Through the genetic analysis of 21 families with early-onset high myopia, this study seeks to contribute to a better understanding of the role of genetics in this disease and to propose candidate genes. Whole-exome sequencing studies with a panel of genes known to be involved in the pathology were performed in families with inconclusive results: 3% of the variants found were classified as pathogenic, 6% were likely pathogenic and the remaining 91% were variants of uncertain significance. Most of the families in this study were found to have alterations in several of the proposed genes. This suggests a polygenic inheritance of the pathology due to the cumulative effect of the alterations. Further studies are needed to validate and confirm the role of these alterations in the development of early-onset high myopia and its polygenic inheritance.
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Miopia , Criança , Humanos , Sequenciamento do Exoma , Miopia/genéticaRESUMO
Pathogenic variants in the AP4B1 gene lead to a rare form of hereditary spastic paraplegia (HSP) known as SPG47. We report on a patient with a clinical suspicion of complicated HSP of the lower limbs with intellectual disability, as well as a novel homozygous noncanonical splice site variant in the AP4B1 gene, in which the effect on splicing was validated by RNA analysis. We sequenced 152 genes associated with HSP using Next-Generation Sequencing (NGS). We isolated total RNA from peripheral blood and generated cDNA using reverse transcription-polymerase chain reaction (RT-PCR). A region of AP4B1 mRNA was amplified by PCR and the fragments obtained were purified from the agarose gel and sequenced. We found a homozygous variant of uncertain significance in the AP4B1 gene NM_006594.4: c.1511-6C>G in the proband. Two different AP4B1 mRNA fragments were obtained in the patient and his carrier parents. The shorter fragment was the predominant fragment in the patient and revealed a deletion with skipping of the AP4B1 exon 10. The patient's longer fragment corresponded to an insertion of the last five nucleotides of AP4B1 intron 9. We confirmed that this variant affects the normal splicing of RNA, sustaining the molecular diagnosis of SPG47 in the patient.
Assuntos
Paraplegia Espástica Hereditária , Complexo 4 de Proteínas Adaptadoras , Subunidades beta do Complexo de Proteínas Adaptadoras , Homozigoto , Humanos , Íntrons , Mutação , Linhagem , RNA , RNA Mensageiro/genética , Paraplegia Espástica Hereditária/genéticaRESUMO
OBJECTIVE: The implementation of pharmacogenetics (PGx) in clinical practice is an essential tool for personalized medicine. However, clinical laboratories must validate their procedures before being used to perform PGx studies in patients, in order to confirm that they are adequate for the intended purposes. METHODS: We designed a validation process for our in-house pharmacogenetic PCR-based method assay. RESULTS: The concordance to reference, repeatability and reproducibility was 100%. Sensitivity and specificity were 100% for the detection of variant diplotypes in CYP2C9, CYP3A5, TPMT, DPYD and UGT1A1 genes. The sensitivity was lower in the detection of CYP2C19 variants due to a limitation in the design that prevents the detection of CYP2C19 *2/*10 diplotype. CONCLUSIONS: The success of implementing clinical pharmacogenetic testing into routine clinical practice is dependent on the precision of genotyping. Limitations must be bearing in mind to guarantee the quality of PGx assays in clinical laboratory practice. We provided objective evidence that the necessary requirements in our laboratory-development assay were fulfilled.
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Laboratórios Clínicos , Farmacogenética , Humanos , Laboratórios , Testes Farmacogenômicos , Reprodutibilidade dos TestesRESUMO
Identifying the druggable target is crucial for patients with nonsquamous advanced non-small cell lung cancer (NSCLC). This article adds to the spectrum of ROS1 fusion cases described in NSCLC. We describe a novel SLC12A2-ROS1 rearrangement that has not been previously reported in other cancers: a fusion that has clinical and radiological sensitivity to crizotinib. Fluorescence in situ hybridization detected the SLC12A2-ROS1 fusion and it was confirmed through hybrid capture-based next-generation sequencing (NGS); however, the fusion could not be detected by amplicon-based assay. The success of implementing NGS into routine clinical practice depends on the accuracy of testing. The test's methodological features should then be considered because they significantly affect the results. Given this patient's response to crizotinib, identifying patients with undescribed ROS1 fusions has important therapeutic implications. KEY POINTS: This is the first known description of an SLC12A2-ROS1 fusion. Considering the patient's clinical features and tumor response observed after crizotinib therapy, the authors confirm that this new rearrangement has relevant clinical impact for patients with non-small cell lung cancer. The success of implementing next-generation sequencing (NGS) into routine clinical practice depends on the accuracy of the testing. Different assays and NGS platforms can achieve differing results. Each assay's limitations need to be considered to ensure the quality of precision medicine in clinical practice.
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Carcinoma Pulmonar de Células não Pequenas , Neoplasias Pulmonares , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Carcinoma Pulmonar de Células não Pequenas/genética , Crizotinibe/farmacologia , Crizotinibe/uso terapêutico , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Hibridização in Situ Fluorescente , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/genética , Proteínas de Fusão Oncogênica/genética , Proteínas Tirosina Quinases/genética , Proteínas Proto-Oncogênicas/genética , Membro 2 da Família 12 de Carreador de SolutoRESUMO
Familial hypercholesterolemia is an autosomal dominant disease of lipid metabolism caused by defects in the genes LDLR, APOB, and PCSK9. The prevalence of heterozygous familial hypercholesterolemia (HeFH) is estimated between 1/200 and 1/250. Early detection of patients with FH allows initiation of treatment, thus reducing the risk of coronary heart disease. In this study, we performed in vitro characterization of new LDLR variants found in our patients. Genetic analysis was performed by Next Generation Sequencing using a customized panel of 198 genes in DNA samples of 516 subjects with a clinical diagnosis of probable or definitive FH. All new LDLR variants found in our patients were functionally validated in CHO-ldlA7 cells. The LDLR activity was measured by flow cytometry and LDLR expression was detected by immunofluorescence. Seven new variants at LDLR were tested: c.518 G>C;p.(Cys173Ser), c.[684 G>T;694 G>T];p.[Glu228Asp;Ala232Ser], c.926C>A;p.(Pro309His), c.1261A>G;p.(Ser421Gly), c.1594T>A;p.(Tyr532Asn), and c.2138delC;p.(Thr713Lysfs*17). We classified all variants as pathogenic except p.(Ser421Gly) and p.(Ala232Ser). The functional in vitro characterization of rare variants at the LDLR is a useful tool to classify the new variants. This approach allows us to confirm the genetic diagnosis of FH, avoiding the classification as "uncertain significant variants", and therefore, carry out cascade family screening.
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Sequenciamento de Nucleotídeos em Larga Escala/métodos , Hiperlipoproteinemia Tipo II/diagnóstico , Mutação , Receptores de LDL/genética , Receptores de LDL/metabolismo , Adolescente , Adulto , Idoso , Animais , Células CHO , Criança , Cricetulus , Diagnóstico Precoce , Feminino , Estudos de Associação Genética , Predisposição Genética para Doença , Humanos , Hiperlipoproteinemia Tipo II/genética , Hiperlipoproteinemia Tipo II/metabolismo , Masculino , Pessoa de Meia-Idade , Análise de Sequência de DNA/métodos , Adulto JovemRESUMO
DUSP6/MKP3 is a dual-specific phosphatase that regulates extracellular regulated kinase ERK1/2 and ERK5 activity, with an increasingly recognized role as tumor suppressor. In silico studies from Gene expression Omnibus (GEO) and Cancer Genome atlas (TCGA) databases reveal poor prognosis in those Non-small cell lung cancer (NSCLC) patients with low expression levels of DUSP6. In agreement with these data, here we show that DUSP6 plays a major role in the regulation of cell migration, motility and tumor growth. We have found upregulation in the expression of several genes involved in epithelial to mesenchymal transition (EMT) in NSCLC-DUSP6 depleted cells. Data obtained in RNA-seq studies carried out in DUSP6 depleted cells identified EGFR, TGF-ß and WNT signaling pathways and several genes such as VAV3, RUNXR2, LEF1, FGFR2 whose expression is upregulated in these cells and therefore affecting cellular functions such as integrin mediated cell adhesion, focal adhesion and motility. Furthermore, EGF signaling pathway is activated via ERK5 and not ERK1/2 and TGF-ß via SMAD2/3 in DUSP6 depleted cells. In summary DUSP6 is a tumor suppressor in NSCLC and re-establishment of its expression may be a potential strategy to revert poor outcome in NSCLC patients.
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Carcinoma Pulmonar de Células não Pequenas/enzimologia , Carcinoma Pulmonar de Células não Pequenas/genética , Fosfatase 6 de Especificidade Dupla/genética , Genes Supressores de Tumor , Neoplasias Pulmonares/enzimologia , Neoplasias Pulmonares/genética , Citoesqueleto de Actina/metabolismo , Adenocarcinoma de Pulmão/enzimologia , Adenocarcinoma de Pulmão/patologia , Junções Aderentes/metabolismo , Animais , Carcinogênese/genética , Carcinogênese/patologia , Carcinoma Pulmonar de Células não Pequenas/patologia , Adesão Celular/genética , Linhagem Celular Tumoral , Movimento Celular/genética , Forma Celular/genética , Progressão da Doença , Fosfatase 6 de Especificidade Dupla/metabolismo , Adesões Focais/metabolismo , Regulação Neoplásica da Expressão Gênica , Humanos , Estimativa de Kaplan-Meier , Neoplasias Pulmonares/patologia , Sistema de Sinalização das MAP Quinases , Camundongos Nus , Fator de Crescimento Transformador beta/metabolismoRESUMO
Glioblastoma (GB) is a devastating tumor of the central nervous system characterized by a poor prognosis. One of the best-established predictive biomarker in IDH-wildtype GB is O6-methylguanine-DNA methyltransferase (MGMT) methylation (mMGMT), which is associated with improved treatment response and survival. However, current efforts to monitor GB patients through mMGMT detection have proven unsuccessful. Small extracellular vesicles (sEVs) hold potential as a key element that could revolutionize clinical practice by offering new possibilities for liquid biopsy. This study aimed to determine the utility of sEV-based liquid biopsy as a predictive biomarker and disease monitoring tool in patients with IDH-wildtype GB. Our findings show consistent results with tissue-based analysis, achieving a remarkable sensitivity of 85.7% for detecting mMGMT in liquid biopsy, the highest reported to date. Moreover, we suggested that liquid biopsy assessment of sEV-DNA could be a powerful tool for monitoring disease progression in IDH-wildtype GB patients. This study highlights the critical significance of overcoming molecular underdetection, which can lead to missed treatment opportunities and misdiagnoses, possibly resulting in ineffective therapies. The outcomes of our research significantly contribute to the field of sEV-DNA-based liquid biopsy, providing valuable insights into tumor tissue heterogeneity and establishing it as a promising tool for detecting GB biomarkers. These results have substantial implications for advancing predictive and therapeutic approaches in the context of GB and warrant further exploration and validation in clinical settings.
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Biomarcadores Tumorais , Neoplasias Encefálicas , Metilação de DNA , Metilases de Modificação do DNA , Enzimas Reparadoras do DNA , Vesículas Extracelulares , Glioblastoma , Proteínas Supressoras de Tumor , Humanos , Glioblastoma/genética , Glioblastoma/patologia , Glioblastoma/diagnóstico , Vesículas Extracelulares/metabolismo , Vesículas Extracelulares/genética , Biópsia Líquida/métodos , Metilases de Modificação do DNA/genética , Metilases de Modificação do DNA/metabolismo , Enzimas Reparadoras do DNA/genética , Enzimas Reparadoras do DNA/metabolismo , Masculino , Feminino , Biomarcadores Tumorais/genética , Biomarcadores Tumorais/metabolismo , Pessoa de Meia-Idade , Proteínas Supressoras de Tumor/genética , Proteínas Supressoras de Tumor/metabolismo , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/patologia , Neoplasias Encefálicas/diagnóstico , Idoso , Adulto , PrognósticoRESUMO
The field of pharmacogenetics (PGx) holds great promise in advancing personalized medicine by adapting treatments based on individual genetic profiles. Despite its benefits, there are still economic, ethical and institutional barriers that hinder its implementation in our healthcare environment. A retrospective analysis approach of anonymized data sourced from electronic health records was performed, encompassing a diverse patient population and evaluating key parameters such as prescribing patterns and test results, to assess the impact of pharmacogenetic testing. A head-to-head comparison with previously published activity results within the same pharmacogenetic laboratory was also conducted to contrast the progress made after 10 years. The analysis revealed significant utilization of pharmacogenetic testing in daily clinical practice, with 1,145 pharmacogenetic tests performed over a 1-year period and showing a 35% growth rate increase over time. Of the 17 different medical departments that sought PGx tests, the Oncology department accounted for the highest number, representing 58.47% of all genotyped patients. A total of 1,000 PGx tests were requested for individuals susceptible to receive a dose modification based on genotype, and 76 individuals received a genotype-guided dose adjustment. This study presents a comprehensive descriptive analysis of real-world data obtained from a public tertiary hospital laboratory specialized in pharmacogenetic testing, and presents data that strongly endorse the integration of pharmacogenetic testing into everyday clinical practice.
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Small extracellular vesicles (sEVs) in the blood of cancer patients contain higher amounts of tumor markers than those identified as free-circulating. miRNAs have significant biomedical relevance due to their high stability and feasible detection. However, there is no reliable endogenous control available to measure sEVs-miRNA content, impairing the acquisition of standardized consistent measurements in cancer liquid biopsy. In this study, we identified three miRNAs from a panel of nine potential normalizers that emerged from a comprehensive analysis comparing the sEV-miRNA profile of six lung and ovarian human cancer cell lines in the absence of or under different conditions. Their relevance as normalizers was tested in 26 additional human cancer cell lines from nine different tumor types undergoing chemotherapy or radiotherapy treatment. The validation cohorts were comprised of 242 prospective plasma and ascitic fluid samples from three different human tumor types. Variability and normalization properties were tested in comparison to miR-16, the most used control to normalize free-circulating miRNAs in plasma. Our results indicate that miR-151a is consistently represented in small extracellular vesicles with minimal variability compared to miR-16, providing a novel normalizer to measure small extracellular vesicle miRNA content that will benefit liquid biopsy in cancer patients.
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BACKGROUND: The diagnosis of cystic fibrosis (CF) is established when characteristic clinical signs are coupled with biallelic CFTR pathogenic variants. No previously reported non-canonical splice site variants have to be considered as variants of uncertain significance unless their effect on splicing has been validated. METHODS: Two variants identified by next-generation sequencing were evaluated. We assayed their effects on splicing employing RNA analysis and real-time expression quantification from RNA obtained from the nasal epithelial cells of a patient with clinically suspected CF and of two patients with milder phenotypes (CFTR-related disorders). RESULTS: The variant c.164+2dup causes skipping of exon 2 (p.(Ser18_Glu54del)) and exon 2 plus 3 (p.(Ser18Argfs*16)) in CFTR mRNA. Exon 2 expression in the patient heterozygous for c.164+2dup was decreased to 7 % of the exon 2 expression in the controls. The synonymous variant c.1584G>A causes a partial skipping of exon 11. The exon 11 expression in the two patients heterozygous for this variant was 22 % and 42 % of that of the controls, respectively. CONCLUSION: We conclude that variant c.164+2dup affects mRNA processing and can be considered a CF-causing variant. The results of the functional assay also showed that the p.(Glu528=) variant, usually categorized as a neutral variant based on epidemiological data, partially affects mRNA processing in our patients. This finding would allow us to reclassify the variant as a CFTR-related variant with incomplete penetrance. RNA obtained from nasal epithelial cells is an easy and accurate tool for CFTR functional studies in patients with unclassified splice variants.
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Introduction: Refractory/relapsed pediatric acute leukemia are still clinically challenging and new therapeutic strategies are needed. Interactions between Natural Killer Group 2D (NKG2D) receptor, expressed in cytotoxic immune cells, and its ligands (NKG2DL), which are upregulated in leukemic blasts, are important for anti-leukemia immunosurveillance. Nevertheless, leukemia cells may develop immunoescape strategies as NKG2DL shedding and/or downregulation. Methods: In this report, we analyzed the anti-leukemia activity of NKG2D chimeric antigen receptor (CAR) redirected memory (CD45RA-) T cells in vitro and in a murine model of T-cell acute lymphoblastic leukemia (T-ALL). We also explored in vitro how soluble NKG2DL (sNKG2DL) affected NKG2D-CAR T cells' cytotoxicity and the impact of NKG2D-CAR T cells on Jurkat cells gene expression and in vivo functionality. Results: In vitro, we found NKG2D-CAR T cells targeted leukemia cells and showed resistance to the immunosuppressive effects exerted by sNKG2DL. In vivo, NKG2D-CAR T cells controlled T cell leukemia burden and increased survival of the treated mice but failed to cure the animals. After CAR T cell treatment, Jurkat cells upregulated genes related to proliferation, survival and stemness, and in vivo, they exhibited functional properties of leukemia initiating cells. Discussion: The data here presented suggest, that, in combination with other therapeutic approaches, NKG2D-CAR T cells could be a novel treatment for pediatric T-ALL.
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Leucemia-Linfoma Linfoblástico de Células T Precursoras , Receptores de Antígenos Quiméricos , Humanos , Criança , Camundongos , Animais , Subfamília K de Receptores Semelhantes a Lectina de Células NK/metabolismo , Leucemia-Linfoma Linfoblástico de Células T Precursoras/terapia , Linhagem Celular Tumoral , Células T de MemóriaRESUMO
BACKGROUND: Two automatable in-house protocols for high-troughput RNA extraction from nasopharyngeal swabs for SARS-CoV-2 detection have been evaluated. METHODS: One hundred forty one SARS-CoV-2 positive samples were collected during a period of 10-days. In-house protocols were based on extraction with magnetic beads and designed to be used with either the Opentrons OT-2 (OT-2in-house) liquid handling robot or the MagMAXTM Express-96 system (MMin-house). Both protocols were tested in parallel with a commercial kit that uses the MagMAXTM system (MMkit). Nucleic acid extraction efficiencies were calculated from a SARS-CoV-2 DNA positive control. RESULTS: No significant differences were found between both in-house protocols and the commercial kit in their performance to detect positive samples. The MMkit was the most efficient although the MMin-house presented, in average, lower Cts than the other two. In-house protocols allowed to save between 350 and 400 for every 96 extracted samples compared to the commercial kit. CONCLUSION: The protocols described harness the use of easily available reagents and an open-source liquid handling system and are suitable for SARS-CoV-2 detection in high throughput facilities.
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Automação Laboratorial/métodos , Teste de Ácido Nucleico para COVID-19/métodos , RNA Viral/normas , Automação Laboratorial/economia , Automação Laboratorial/normas , Teste de Ácido Nucleico para COVID-19/economia , Teste de Ácido Nucleico para COVID-19/normas , Custos e Análise de Custo , Humanos , RNA Viral/química , RNA Viral/genética , Kit de Reagentes para Diagnóstico/economia , Kit de Reagentes para Diagnóstico/normas , Sensibilidade e EspecificidadeRESUMO
BACKGROUND: In an effort to contribute to overcoming the platinum resistance exhibited by most solid tumors, we performed an array of epigenetic approaches, integrating next-generation methodologies and public clinical data to identify new potential epi-biomarkers in ovarian cancer, which is considered the most devastating of gynecological malignancies. METHODS: We cross-analyzed data from methylome assessments and restoration of gene expression through microarray expression in a panel of four paired cisplatin-sensitive/cisplatin-resistant ovarian cancer cell lines, along with publicly available clinical data from selected individuals representing the state of chemoresistance. We validated the methylation state and expression levels of candidate genes in each cellular phenotype through Sanger sequencing and reverse transcription polymerase chain reaction, respectively. We tested the biological role of selected targets using an ectopic expression plasmid assay in the sensitive/resistant tumor cell lines, assessing the cell viability in the transfected groups. Epigenetic features were also assessed in 189 primary samples obtained from ovarian tumors and controls. RESULTS: We identified PAX9 and FKBP1B as potential candidate genes, which exhibited epigenetic patterns of expression regulation in the experimental approach. Re-establishment of FKBP1B expression in the resistant OVCAR3 phenotype in which this gene is hypermethylated and inhibited allowed it to achieve a degree of platinum sensitivity similar to the sensitive phenotype. The evaluation of these genes at a translational level revealed that PAX9 hypermethylation leads to a poorer prognosis in terms of overall survival. We also set a precedent for establishing a common epigenetic signature in which the validation of a single candidate, MEST, proved the accuracy of our computational pipelines. CONCLUSIONS: Epigenetic regulation of PAX9 and FKBP1B genes shows that methylation in non-promoter areas has the potential to control gene expression and thus biological consequences, such as the loss of platinum sensitivity. At the translational level, PAX9 behaves as a predictor of chemotherapy response to platinum in patients with ovarian cancer. This study revealed the importance of the transcript-specific study of each gene under potential epigenetic regulation, which would favor the identification of new markers capable of predicting each patient's progression and therapeutic response.
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Proteínas Adaptadoras de Transdução de Sinal/genética , Carcinoma Epitelial do Ovário/tratamento farmacológico , Metilação de DNA/efeitos dos fármacos , Epigênese Genética/efeitos dos fármacos , Neoplasias Ovarianas/tratamento farmacológico , Fator de Transcrição PAX9/genética , Compostos de Platina/uso terapêutico , Adulto , Idoso , Idoso de 80 Anos ou mais , Antineoplásicos/uso terapêutico , Linhagem Celular Tumoral/efeitos dos fármacos , Cisplatino/uso terapêutico , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Feminino , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Variação Genética , Humanos , Pessoa de Meia-Idade , EspanhaRESUMO
X-linked myotubular myopathy (XLMTM; OMIM 310400) is a centronuclear congenital muscular disorder of X-linked recessive inheritance. Although female carriers are typically asymptomatic, affected heterozygous females have been described. Here, we describe the case of a sporadic female patient with suspicion of centronuclear myopathy and a heterozygous large deletion at Xq28 encompassing the MAMLD1, MTM1, MTMR1, CD99L2, and HMGB3 genes. The deletion was first detected using a custom next generation sequencing (NGS)-based multigene panel and finally characterized by comparative genomic hybridization array and multiplex ligation probe assay techniques. In this patient we have confirmed, by MTM1 mRNA quantification, a MTM1 gene expression less than the expected 50 percent in patient muscle. The significant 20% reduction in MTM1 mRNA expression in muscle, precludes low level of the normal myotubularin protein as the cause of the phenotype in this heterozygous female. We have also found that BIN1 expression in patient muscle biopsy was significantly increased, and postulate that BIN1 expression will be increased in XLMTM patient muscle as an attempt to maintain muscle function.
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Proteínas Adaptadoras de Transdução de Sinal/genética , Deleção Cromossômica , Miopatias Congênitas Estruturais/genética , Proteínas Nucleares/genética , Proteínas Tirosina Fosfatases não Receptoras/genética , Proteínas Supressoras de Tumor/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Adolescente , Cromossomos Humanos X/genética , Feminino , Heterozigoto , Humanos , Músculo Esquelético/metabolismo , Músculo Esquelético/patologia , Miopatias Congênitas Estruturais/metabolismo , Miopatias Congênitas Estruturais/patologia , Proteínas Nucleares/metabolismo , Proteínas Tirosina Fosfatases não Receptoras/metabolismo , Proteínas Supressoras de Tumor/metabolismoRESUMO
BACKGROUND: The promoter hypermethylation of the methylguanine-DNA methyltransferase gene is a frequently used biomarker in daily clinical practice as it is associated with a favorable prognosis in glioblastoma patients treated with temozolamide. Due to the absence of adequately standardized techniques, international harmonization of the MGMT methylation biomarker is still an unmet clinical need for the diagnosis and treatment of glioblastoma patients. RESULTS: In this study we carried out a clinical validation of a quantitative assay for MGMT methylation detection by comparing a novel quantitative MSP using double-probe (dp_qMSP) with the conventional MSP in 100 FFPE glioblastoma samples. We performed both technologies and established the best cutoff for the identification of positive-methylated samples using the quantitative data obtained from dp_qMSP. Kaplan-Meier curves and ROC time dependent curves were employed for the comparison of both methodologies. CONCLUSIONS: We obtained similar results using both assays in the same cohort of patients, in terms of progression free survival and overall survival according to Kaplan-Meier curves. In addition, the results of ROC(t) curves showed that dp_qMSP increases the area under curve time-dependent in comparison with MSP for predicting progression free survival and overall survival over time. We concluded that dp_qMSP is an alternative methodology compatible with the results obtained with the conventional MSP. Our assay will improve the therapeutic management of glioblastoma patients, being a more sensitive and competitive alternative methodology that ensures the standardization of the MGMT-biomarker making it reliable and suitable for clinical use.
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Biomarcadores Tumorais/genética , Neoplasias Encefálicas/patologia , Glioblastoma/diagnóstico , Glioblastoma/genética , Adulto , Idoso , Idoso de 80 Anos ou mais , Antineoplásicos Alquilantes/uso terapêutico , Neoplasias Encefálicas/mortalidade , Estudos de Coortes , Ilhas de CpG , Metilação de DNA , Metilases de Modificação do DNA/genética , Enzimas Reparadoras do DNA/genética , Epigenômica , Feminino , Glioblastoma/tratamento farmacológico , Glioblastoma/cirurgia , Humanos , Estimativa de Kaplan-Meier , Masculino , Pessoa de Meia-Idade , Reação em Cadeia da Polimerase/métodos , Reação em Cadeia da Polimerase/tendências , Prognóstico , Intervalo Livre de Progressão , Regiões Promotoras Genéticas/genética , Estudos Prospectivos , Curva ROC , Sensibilidade e Especificidade , Temozolomida/uso terapêutico , Proteínas Supressoras de Tumor/genéticaRESUMO
Natural killer (NK) cells represent promising tools for cancer immunotherapy. We report the optimization of an NK cell activation-expansion process and its validation on clinical-scale. METHODS: RPMI-1640, stem cell growth medium (SCGM), NK MACS and TexMACS were used as culture mediums. Activated and expanded NK cells (NKAE) were obtained by coculturing total peripheral blood mononuclear cells (PBMC) or CD45RA+ cells with irradiated K562mbIL15-41BBL or K562mbIL21-41BBL. Fold increase, NK cell purity, activation status, cytotoxicity and transcriptome profile were analyzed. Clinical-grade NKAE cells were manufactured in CliniMACS Prodigy. RESULTS: NK MACS and TexMACs achieved the highest NK cell purity and lowest T cell contamination. Obtaining NKAE cells from CD45RA+ cells was feasible although PBMC yielded higher total cell numbers and NK cell purity than CD45RA+ cells. The highest fold expansion and NK purity were achieved by using PBMC and K562mbIL21-41BBL cells. However, no differences in activation and cytotoxicity were found when using either NK cell source or activating cell line. Transcriptome profile showed to be different between basal NK cells and NKAE cells expanded with K562mbIL21-41BBL or K562mbIL15-41BBL. Clinical-grade manufactured NKAE cells complied with the specifications from the Spanish Regulatory Agency. CONCLUSIONS: GMP-grade NK cells for clinical use can be obtained by using different starting cells and aAPC.
RESUMO
BACKGROUND: Cytosine modifications in DNA such as 5-methylcytosine (5mC) underlie a broad range of developmental processes, maintain cellular lineage specification, and can define or stratify types of cancer and other diseases. However, the wide variety of approaches available to interrogate these modifications has created a need for harmonized materials, methods, and rigorous benchmarking to improve genome-wide methylome sequencing applications in clinical and basic research. Here, we present a multi-platform assessment and cross-validated resource for epigenetics research from the FDA's Epigenomics Quality Control Group. RESULTS: Each sample is processed in multiple replicates by three whole-genome bisulfite sequencing (WGBS) protocols (TruSeq DNA methylation, Accel-NGS MethylSeq, and SPLAT), oxidative bisulfite sequencing (TrueMethyl), enzymatic deamination method (EMSeq), targeted methylation sequencing (Illumina Methyl Capture EPIC), single-molecule long-read nanopore sequencing from Oxford Nanopore Technologies, and 850k Illumina methylation arrays. After rigorous quality assessment and comparison to Illumina EPIC methylation microarrays and testing on a range of algorithms (Bismark, BitmapperBS, bwa-meth, and BitMapperBS), we find overall high concordance between assays, but also differences in efficiency of read mapping, CpG capture, coverage, and platform performance, and variable performance across 26 microarray normalization algorithms. CONCLUSIONS: The data provided herein can guide the use of these DNA reference materials in epigenomics research, as well as provide best practices for experimental design in future studies. By leveraging seven human cell lines that are designated as publicly available reference materials, these data can be used as a baseline to advance epigenomics research.
Assuntos
Epigênese Genética , Epigenômica/métodos , Controle de Qualidade , 5-Metilcitosina , Algoritmos , Ilhas de CpG , DNA/genética , Metilação de DNA , Epigenoma , Genoma Humano , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Alinhamento de Sequência , Análise de Sequência de DNA/métodos , Sulfitos , Sequenciamento Completo do Genoma/métodosRESUMO
Deep learning has enabled great advances to be made in cancer research with regards to diagnosis, prognosis, and treatment. The study by Wang and colleagues in this issue of Cancer Research develops a deep learning algorithm with the ability to digitally stain histologic images, achieving reliable nuclei segmentation and cell classification. They use this tool to study the tumor morphologic microenvironment in tissue pathology images of patients with lung adenocarcinoma. On the basis of the image features, they develop a prognostic model and find correlations with the transcriptional activities of biological pathways.See related article by Wang et al., p. 2056.