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1.
Pediatr Blood Cancer ; 62(8): 1368-73, 2015 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-25939774

RESUMO

BACKGROUND: The clinical significance of minimal residual disease (MRD) detected by real-time quantitative PCR (qPCR) in autologous stem cell grafts in high risk neuroblastoma is still controversial. In this retrospective multicenter study, autologous stem cell grafts of a large cohort were studied using a panel of RNA markers. PROCEDURE: From 104 patients with high risk neuroblastoma, who received autologous stem cell transplantation as first line treatment, 66 peripheral blood stem cells (PBSC) and 38 CD34+ selected grafts were retrospectively collected at 2 Dutch and 12 German centers between 1997 and 2010. To investigate graft contamination qPCR was performed by using 5 neuroblastoma specific markers (PHOX2B, TH, DDC, CHRNA3, and DBH). RESULTS: In PBSC 6/66 (9%) and in CD34+ selected grafts 3/38 (8%) samples were contaminated. Graft contamination was not associated with an unfavorable outcome (5-years OS, 66% vs. 50.5%; P=0.6 and 5-years EFS, 22% vs. 35%, P=0.7). In multivariate Cox analysis BM MRD at time of harvest was significantly associated with survival (P=0.008 OS and P=0.002 EFS), but graft contamination was still not associated with an unfavorable outcome (P=0.9 OS and P=1 EFS). CONCLUSIONS: Graft contamination is very infrequent in this retrospective cohort of patients with no or minimal BM disease prior to stem cell collection and does not influence outcome in univariate and multivariate analysis. The presence of MRD at time of harvest is a strong outcome predictor. However, these results will have to be verified in a large prospective study.


Assuntos
Neoplasia Residual/patologia , Células Neoplásicas Circulantes/patologia , Neuroblastoma/patologia , Transplante de Células-Tronco , Adolescente , Descarboxilases de Aminoácido-L-Aromático/genética , Criança , Pré-Escolar , Proteínas de Homeodomínio/genética , Humanos , Lactente , Reação em Cadeia da Polimerase em Tempo Real , Receptores Nicotínicos/genética , Estudos Retrospectivos , Fatores de Transcrição/genética , Transplante Autólogo
2.
J Sci Food Agric ; 92(2): 439-44, 2012 Jan 30.
Artigo em Inglês | MEDLINE | ID: mdl-21968969

RESUMO

BACKGROUND: The small intestinal epithelium functions both to absorb nutrients, and to provide a barrier between the outside, luminal, world and the human body. One of the passageways across the intestinal epithelium is paracellular diffusion, which is controlled by the properties of tight junction complexes. We used a differentiated Caco-2 monolayer as a model for small intestinal epithelium to study the effect of crude apple extracts on paracellular permeability. RESULTS: Exposure of crude apple homogenate to the differentiated Caco-2 cells increased the paracellular resistance, determined as trans-epithelial electrical resistance (TEER). This increase was linearly related to the concentration of apple present. The TEER-enhancing effect of apple extract was due to factors mainly present in the cortex, and the induction was not inhibited by protein kinase inhibitors. Apple-induced resistance was accompanied by increased expression of several tight junction related genes, including claudin 4 (CLDN4). CONCLUSION: Crude apple extract induces a higher paracellular resistance in differentiated Caco-2 cells. Future research will determine whether these results can be extrapolated to human small intestinal epithelia.


Assuntos
Claudinas/metabolismo , Células Epiteliais/efeitos dos fármacos , Regulação da Expressão Gênica/efeitos dos fármacos , Malus/química , Extratos Vegetais/farmacologia , Células CACO-2 , Claudina-4 , Claudinas/genética , Relação Dose-Resposta a Droga , Frutas/química , Perfilação da Expressão Gênica , Humanos , Extratos Vegetais/administração & dosagem , Extratos Vegetais/química , Junções Íntimas/fisiologia
3.
J Crit Care ; 64: 219-225, 2021 08.
Artigo em Inglês | MEDLINE | ID: mdl-34023553

RESUMO

BACKGROUND: Prolonged viral RNA detection in respiratory samples from patients with COVID-19 has been described, but the clinical relevance remains unclear. We studied the dynamics of SARS-CoV-2 on a group and individual level in intubated ICU patients. METHODS: In a cohort of 86 patients, we analysed SARS-CoV-2 RT-PCR results on nasopharyngeal and sputum samples (obtained as part of clinical care twice a week) according to time after intubation. Subsequently, we performed survival analyses. RESULTS: 870 samples were tested by RT-PCR. Overall viral load was highest in the first week (median nasopharynx 3.5, IQR 1.5-4.3; median sputum 4.3, IQR 3.3-5.6) and decreased over time. In 20% of patients a relapsing pattern was observed. Nasopharyngeal and sputum PCR status on day 14 was not significantly associated with survival up to day 60 in this small cohort. CONCLUSION: In general SARS-CoV-2 RNA levels in respiratory samples in patients with severe COVID-19 decrease after the first week after intubation, but individual SARS-CoV-2 RNA levels can show a relapsing pattern. Larger studies are needed to address the association of clearance of SARS-CoV-2 RNA from respiratory samples with survival, because we observed a trend towards better survival in patients with early clearance from sputum.


Assuntos
Teste de Ácido Nucleico para COVID-19 , COVID-19/diagnóstico , COVID-19/virologia , RNA Viral , SARS-CoV-2 , Carga Viral , Idoso , Feminino , Humanos , Unidades de Terapia Intensiva , Intubação , Masculino , Pessoa de Meia-Idade , Nasofaringe/virologia , Países Baixos/epidemiologia , Escarro/virologia , Análise de Sobrevida
4.
Artigo em Inglês | MEDLINE | ID: mdl-32923888

RESUMO

PURPOSE: Circulating tumor DNA (ctDNA) has been used for disease monitoring in several types of cancer. The aim of our study was to investigate whether ctDNA can be used for response monitoring in neuroblastoma. METHODS: One hundred forty-nine plasma samples from 56 patients were analyzed by quantitative polymerase chain reaction (qPCR) for total cell free DNA (cfDNA; albumin and ß-actin) and ctDNA (hypermethylated RASSF1A). ctDNA results were compared with mRNA-based minimal residual disease (qPCR) in bone marrow (BM) and blood and clinical patient characteristics. RESULTS: ctDNA was detected at diagnosis in all patients with high-risk and stage M neuroblastoma and in 3 of 7 patients with localized disease. The levels of ctDNA were highest at diagnosis, decreased during induction therapy, and not detected before or after autologous stem-cell transplantation. At relapse, the amount of ctDNA was comparable to levels at diagnosis. There was an association between ctDNA and blood or BM mRNA, with concordant results when tumor burden was high or no tumor was detected. The discrepancies indicated either low-level BM infiltration (ctDNA negative/mRNA positive) or primary tumor/soft tissue lesions with no BM involvement (ctDNA positive/mRNA negative). CONCLUSION: ctDNA can be used for monitoring disease in patients with neuroblastoma. In high-risk patients and all patients with stage M at diagnosis, ctDNA is present. Our data indicate that at low tumor load, testing of both ctDNA and mRNA increases the sensitivity of molecular disease monitoring. It is likely that ctDNA can originate from both primary tumor and metastases and may be of special interest for disease monitoring in patients who experience relapse in other organs than BM.

5.
Artigo em Inglês | MEDLINE | ID: mdl-34036221

RESUMO

Patients with neuroblastoma in molecular remission remain at considerable risk for disease recurrence. Studies have found that neuroblastoma tissue contains adrenergic (ADRN) and mesenchymal (MES) cells; the latter express low levels of commonly used markers for minimal residual disease (MRD). We identified MES-specific MRD markers and studied the dynamics of these markers during treatment. PATIENTS AND METHODS: Microarray data were used to identify genes differentially expressed between ADRN and MES cell lines. Candidate genes were then studied using real-time quantitative polymerase chain reaction in cell lines and control bone marrow and peripheral blood samples. After selecting a panel of markers, serial bone marrow, peripheral blood, and peripheral blood stem cell samples were obtained from patients with high-risk neuroblastoma and tested for marker expression; survival analyses were also performed. RESULTS: PRRX1, POSTN, and FMO3 mRNAs were used as a panel for specifically detecting MES mRNA in patient samples. MES mRNA was detected only rarely in peripheral blood; moreover, the presence of MES mRNA in peripheral blood stem cell samples was associated with low event-free survival and overall survival. Of note, during treatment, serial bone marrow samples obtained from 29 patients revealed a difference in dynamics between MES mRNA markers and ADRN mRNA markers. Furthermore, MES mRNA was detected in a higher percentage of patients with recurrent disease than in those who remained disease free (53% v 32%, respectively; P = .03). CONCLUSION: We propose that the markers POSTN and PRRX1, in combination with FMO3, be used for real-time quantitative polymerase chain reaction-based detection of MES neuroblastoma mRNA in patient samples because these markers have a unique pattern during treatment and are more prevalent in patients with poor outcome. Together with existing markers of MRD, these new markers should be investigated further in large prospective studies.

7.
Eur J Cancer ; 54: 149-158, 2016 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-26796600

RESUMO

INTRODUCTION: The clinical importance of the detection of neuroblastoma messenger RNA (mRNA) in bone marrow (BM) of localised neuroblastoma patients at diagnosis remains unclear. In this prospective multicentre study, BM samples of a large cohort, were studied using real-time quantitative polymerase chain reaction (qPCR). METHODS: BM samples at diagnosis from 160 patients with localised neuroblastoma were prospectively collected at Dutch and German centres between 2009 and 2013. qPCR was performed using five neuroblastoma specific markers. The association with other biological factors and the prognostic impact of BM positivity and clinical response was assessed. RESULTS: In 58 out of 160 patients neuroblastoma mRNA was detected in BM. In 47 of the 58 positive samples only one marker was found positive. BM positivity was significantly associated with MYCN amplification (p = 0.02) and deletion of chromosome 1p (p = 0.04). In total 31 patients had an event, of which only five patients had progression to stage IV. BM positivity was not associated with an unfavourable outcome. However, the detection of more than one marker was associated with an unfavourable outcome (systemic or local relapse) (event free survival 48% versus 85%; p = 0.03) in the whole cohort and in the observation group. CONCLUSIONS: BM positivity was associated with unfavourable biological factors and might represent more aggressive tumours. Patients with qPCR positive BM should not be upstaged, because of very few systemic events in the cohort. However, for patients with more than one marker positive a more careful follow-up is advisable. These results need to be verified in a very large cohort of localised patients.


Assuntos
Biomarcadores Tumorais/genética , Medula Óssea/química , Neuroblastoma/genética , RNA Mensageiro/genética , RNA Neoplásico/genética , Adolescente , Exame de Medula Óssea , Criança , Pré-Escolar , Deleção Cromossômica , Cromossomos Humanos Par 1 , Progressão da Doença , Intervalo Livre de Doença , Feminino , Amplificação de Genes , Predisposição Genética para Doença , Alemanha , Humanos , Lactente , Recém-Nascido , Estimativa de Kaplan-Meier , Masculino , Proteína Proto-Oncogênica N-Myc , Recidiva Local de Neoplasia , Estadiamento de Neoplasias , Países Baixos , Neuroblastoma/mortalidade , Neuroblastoma/secundário , Neuroblastoma/terapia , Proteínas Nucleares/genética , Proteínas Oncogênicas/genética , Fenótipo , Valor Preditivo dos Testes , Estudos Prospectivos , Reação em Cadeia da Polimerase em Tempo Real , Fatores de Risco , Fatores de Tempo , Resultado do Tratamento , Adulto Jovem
8.
J Mol Diagn ; 17(1): 43-52, 2015 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-25445214

RESUMO

PCR-based detection of minimal residual disease (MRD) in neuroblastoma is currently based on RNA markers; however, expression of these targets can vary, and only paired-like homeobox 2b has no background expression. We investigated whether chromosomal breakpoints, identified by whole-genome sequencing (WGS), can be used as patient-specific DNA MRD markers. WGS data were used to develop large numbers of real-time PCRs specific for tumors of eight patients. These PCRs were used to quantify chromosomal breakpoints in primary tumor and bone marrow samples. Finally, the DNA breakpoints with the highest abundance were compared with a panel of RNA markers. By WGS we identified 42 chromosomal breakpoints in tumor samples from eight patients and developed specific quantitative real-time PCRs for each breakpoint. The tumor-specific breakpoints were all present in bone marrow at diagnosis. For one patient slight clonal selection was observed in response to treatment. Positivity of DNA MRD markers preceded disease progression in four of five patients; in one patient the RNA markers remained negative. For 16 of 22 samples MRD levels determined by RNA and DNA were comparable and in 6 of 22 samples higher MRD levels were detected by DNA markers. DNA breakpoints used as MRD targets in neuroblastoma are reliable and stable markers. In addition, this technique might be applicable for detecting tumor cells in other types of cancer.


Assuntos
Biomarcadores Tumorais/genética , Pontos de Quebra do Cromossomo , Neoplasia Residual/genética , Neuroblastoma/genética , Reação em Cadeia da Polimerase em Tempo Real/métodos , Antineoplásicos/uso terapêutico , Medula Óssea/metabolismo , Medula Óssea/patologia , Criança , Pré-Escolar , Feminino , Marcadores Genéticos , Genoma Humano , Estudo de Associação Genômica Ampla , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Lactente , Masculino , Estadiamento de Neoplasias , Neoplasia Residual/tratamento farmacológico , Neoplasia Residual/mortalidade , Neoplasia Residual/patologia , Neuroblastoma/tratamento farmacológico , Neuroblastoma/mortalidade , Neuroblastoma/patologia , Análise de Sobrevida
9.
Nat Genet ; 47(8): 864-71, 2015 08.
Artigo em Inglês | MEDLINE | ID: mdl-26121087

RESUMO

The majority of patients with neuroblastoma have tumors that initially respond to chemotherapy, but a large proportion will experience therapy-resistant relapses. The molecular basis of this aggressive phenotype is unknown. Whole-genome sequencing of 23 paired diagnostic and relapse neuroblastomas showed clonal evolution from the diagnostic tumor, with a median of 29 somatic mutations unique to the relapse sample. Eighteen of the 23 relapse tumors (78%) showed mutations predicted to activate the RAS-MAPK pathway. Seven of these events were detected only in the relapse tumor, whereas the others showed clonal enrichment. In neuroblastoma cell lines, we also detected a high frequency of activating mutations in the RAS-MAPK pathway (11/18; 61%), and these lesions predicted sensitivity to MEK inhibition in vitro and in vivo. Our findings provide a rationale for genetic characterization of relapse neuroblastomas and show that RAS-MAPK pathway mutations may function as a biomarker for new therapeutic approaches to refractory disease.


Assuntos
Sistema de Sinalização das MAP Quinases/genética , Proteínas Quinases Ativadas por Mitógeno/genética , Mutação , Recidiva Local de Neoplasia/genética , Neuroblastoma/genética , Proteínas ras/genética , Quinase do Linfoma Anaplásico , Animais , Benzimidazóis/farmacologia , Western Blotting , Linhagem Celular Tumoral , Criança , Pré-Escolar , Aberrações Cromossômicas , Inibidor p16 de Quinase Dependente de Ciclina/genética , Inibidor p16 de Quinase Dependente de Ciclina/metabolismo , Feminino , Regulação Neoplásica da Expressão Gênica , Células HEK293 , Humanos , Lactente , Masculino , Camundongos SCID , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Neuroblastoma/tratamento farmacológico , Neuroblastoma/patologia , Fosforilação/efeitos dos fármacos , Receptores Proteína Tirosina Quinases/genética , Receptores Proteína Tirosina Quinases/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Ensaios Antitumorais Modelo de Xenoenxerto , Proteínas ras/metabolismo
10.
Ned Tijdschr Geneeskd ; 156(51): A4677, 2012.
Artigo em Holandês | MEDLINE | ID: mdl-23249505

RESUMO

We describe two children who presented with extreme weight gain and failure to grow in height, and who were both diagnosed with Cushing's disease. Following preoperative metyrapone treatment, both children successfully underwent curative transsphenoidal surgery. While obesity is a common and increasing problem in childhood, Cushing's disease is rare. The combination of weight gain and growth failure in a child is an alarming sign in which underlying endocrine disease must be ruled out.


Assuntos
Estatura/fisiologia , Síndrome de Cushing/diagnóstico , Obesidade/diagnóstico , Adenoma/sangue , Adenoma/diagnóstico , Adenoma/cirurgia , Criança , Síndrome de Cushing/sangue , Síndrome de Cushing/cirurgia , Dexametasona , Diagnóstico Diferencial , Feminino , Humanos , Neoplasias Hipofisárias/sangue , Neoplasias Hipofisárias/diagnóstico , Neoplasias Hipofisárias/cirurgia , Resultado do Tratamento
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