Your browser doesn't support javascript.
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 51
Filtrar
Mais filtros










Base de dados
Intervalo de ano de publicação
1.
Cells ; 8(12)2019 12 14.
Artigo em Inglês | MEDLINE | ID: mdl-31847394

RESUMO

Liquid biopsy-based biomarkers, such as microRNAs, represent valuable tools for patient management, but often do not make it to integration in the clinic. We aim to explore issues impeding this transition, in the setting of germ cell tumors, for which novel biomarkers are needed. We describe a model for identifying and validating clinically relevant microRNAs for germ cell tumor patients, using both in vitro, in vivo (mouse model) and patient-derived data. Initial wide screening of candidate microRNAs is performed, followed by targeted profiling of potentially relevant biomarkers. We demonstrate the relevance of appropriate (negative) controls, experimental conditions (proliferation), and issues related to sample origin (serum, plasma, cerebral spinal fluid) and pre-analytical variables (hemolysis, contaminants, temperature), all of which could interfere with liquid biopsy-based studies and their conclusions. Finally, we show the value of our identification model in a specific scenario, contradicting the presumed role of miR-375 as marker of teratoma histology in liquid biopsy setting. Our findings indicate other putative microRNAs (miR-885-5p, miR-448 and miR-197-3p) fulfilling this clinical need. The identification model is informative to identify the best candidate microRNAs to pursue in a clinical setting.

2.
Cancers (Basel) ; 11(9)2019 Sep 17.
Artigo em Inglês | MEDLINE | ID: mdl-31533343

RESUMO

BACKGROUND: The event of X chromosome inactivation induced by XIST, which is physiologically observed in females, is retained in testicular germ cell tumors (TGCTs), as a result of a supernumerary X chromosome constitution. X chromosome inactivation also occurs in male germline, specifically during spermatogenesis. We aimed to analyze the promoter methylation status of XIST in a series of TGCT tissues, representative cell lines, and testicular parenchyma. METHODS: Two independent cohorts were included, comprising a total of 413 TGCT samples, four (T)GCT cell lines, and 86 testicular parenchyma samples. The relative amount of methylated and demethylated XIST promoter fragments was assessed by quantitative methylation-specific PCR (qMSP) and more sensitive high-resolution melting (HRM) methylation analyses. RESULTS: Seminomas showed a lower amount of methylated XIST fragments as compared to non-seminomas or normal testis (p < 0.0001), allowing for a good discrimination among these groups (area under the curve 0.83 and 0.81, respectively). Seminomas showed a significantly higher content of demethylated XIST as compared to non-seminomas. The percentage of demethylated XIST fragment in cell lines reflected their chromosomal constitution (number of extra X chromosomes). A novel and strong positive correlation between the Johnsen's score and XIST demethylation was identified (r = 0.75, p < 0.0001). CONCLUSIONS: The X chromosome inactivation event and demethylated XIST promoter are promising biomarkers for TGCTs and for assessing spermatogenesis quality.

3.
Am J Surg Pathol ; 43(12): 1711-1719, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31490238

RESUMO

Vascular invasion has been identified as an informative risk factor for relapse in stage I testicular nonseminomas, used to tailor treatment. We investigated interobserver agreement in vascular invasion reporting and studied the potential additional value of immunohistochemistry for vascular markers for predicting relapse. Patients (n=52) with stage I testicular nonseminomas undergoing surveillance (1993-2006) were included (median follow-up of 66 mo). Two formalin-fixed paraffin-embedded blocks with >1 cm tissue and tumor/normal parenchyma interface were stained with hematoxylin and eosin and CD31, FVIII, and D2-40. Slides were assessed by 3 independent testicular germ cell tumor-dedicated pathologists, and agreement was assessed using Cohen κ statistic. Sensitivity, specificity, and accuracy of vascular invasion scoring in predicting relapse were calculated. Agreement among testicular germ cell tumor-dedicated pathologists was moderate (κ=0.49 to 0.54), as was performance in predicting disease relapse (particularly, specificity of 86%). Immunohistochemistry increased overall sensitivity (71%), but decreased specificity (71%) in predicting relapse. All patients (n=8) with both blood and lymphatic vascular invasion developed a relapse. In multivariable analysis (including age, tumor size, rete testis invasion, and serum tumor markers), only vascular invasion had an independent impact in predicting relapse. Assessment of vascular invasion by testicular germ cell tumor-dedicated pathologists is good and is clinically meaningful, predicting disease relapse. Immunohistochemistry for vascular markers improves sensitivity of detecting disease relapse and allows for the identification of high-risk patients with both blood and lymphatic vascular invasion simultaneously, potentially of interest for tailored chemotherapy.

4.
Br J Cancer ; 120(4): 444-452, 2019 02.
Artigo em Inglês | MEDLINE | ID: mdl-30739914

RESUMO

BACKGROUND: Testicular germ cell cancer (TGCC), being the most frequent malignancy in young Caucasian males, is initiated from an embryonic germ cell. This study determines intratumour heterogeneity to unravel tumour progression from initiation until metastasis. METHODS: In total, 42 purified samples of four treatment-resistant nonseminomatous (NS) TGCC were investigated, including the precursor germ cell neoplasia in situ (GCNIS) and metastatic specimens, using whole-genome and targeted sequencing. Their evolution was reconstructed. RESULTS: Intratumour molecular heterogeneity did not correspond to the supposed primary tumour histological evolution. Metastases after systemic treatment could be derived from cancer stem cells not identified in the primary cancer. GCNIS mostly lacked the molecular marks of the primary NS and comprised dominant clones that failed to progress. A BRCA-like mutational signature was observed without evidence for direct involvement of BRCA1 and BRCA2 genes. CONCLUSIONS: Our data strongly support the hypothesis that NS is initiated by whole-genome duplication, followed by chromosome copy number alterations in the cancer stem cell population, and accumulation of low numbers of somatic mutations, even in therapy-resistant cases. These observations of heterogeneity at all stages of tumourigenesis should be considered when treating patients with GCNIS-only disease, or with clinically overt NS.


Assuntos
Neoplasias Embrionárias de Células Germinativas/genética , Neoplasias Testiculares/genética , Evolução Molecular , Genes BRCA1 , Genes BRCA2 , Humanos , Perda de Heterozigosidade , Masculino , Mutação , Metástase Neoplásica , Neoplasias Embrionárias de Células Germinativas/patologia , Neoplasias Testiculares/patologia , Sequenciamento Completo do Genoma
5.
Int J Mol Sci ; 20(2)2019 Jan 10.
Artigo em Inglês | MEDLINE | ID: mdl-30634670

RESUMO

Current (high throughput omics-based) data support the model that human (malignant) germ cell tumors are not initiated by somatic mutations, but, instead through a defined locked epigenetic status, representative of their cell of origin. This elegantly explains the role of both genetic susceptibility as well as environmental factors in the pathogenesis, referred to as 'genvironment'. Moreover, it could also explain various epidemiological findings, including the rising incidence of this type of cancer in Western societies. In addition, it allows for identification of clinically relevant and informative biomarkers both for diagnosis and follow-up of individual patients. The current status of these findings will be discussed, including the use of high throughput DNA methylation profiling for determination of differentially methylated regions (DMRs) as well as chromosomal copy number variation (CNV). Finally, the potential value of methylation-specific tumor DNA fragments (i.e., XIST promotor) as well as embryonic microRNAs as molecular biomarkers for cancer detection in liquid biopsies will be presented.


Assuntos
Transformação Celular Neoplásica/genética , Epigênese Genética , Neoplasias Embrionárias de Células Germinativas/etiologia , Neoplasias Embrionárias de Células Germinativas/patologia , Animais , Biomarcadores Tumorais , Meio Ambiente , Interação Gene-Ambiente , Predisposição Genética para Doença , Humanos , Neoplasias Embrionárias de Células Germinativas/epidemiologia
6.
Transplantation ; 103(2): 329-335, 2019 02.
Artigo em Inglês | MEDLINE | ID: mdl-30444806

RESUMO

BACKGROUND: Delayed graft function (DGF), a common complication after transplantation of deceased donor kidneys, affects both short- and long-term outcomes. Currently available biomarkers during graft preservation lack sensitivity in predicting risk for DGF. The aim of this study is to identify cell-free micro ribonucleic acid (miRNA) biomarkers in graft preservation fluid predictive of DGF after kidney transplantation. METHODS: Vascular bed preservation fluid was collected from 48 kidney grafts from donation after circulatory death (DCD) or donation after brain death (DBD) donors. miRNA profiles were determined by polymerase chain reaction (PCR) array (n = 8) and validated by reverse transcription and quantitative PCR (n = 40). Graft function posttransplantation was defined as immediate good function (IF) or DGF. RESULTS: A total of 223 miRNAs fulfilled the preset parameters (Ct < 40 in 3 or more samples) and were included in the analysis. Thirty-two miRNAs were significantly different between DGF and IF kidney grafts (P < 0.05) but, after correction for multiple testing, only miR-505-3p remained significant. The significant association of high miR-505-3p levels with DGF was confirmed in an independent validation cohort using conventional reverse transcription and quantitative PCR detection. Multivariate analyses showed miR-505-3p as an independent predictor for DGF (odds ratio, 1.12; P = 0.028). If stratified for donor type, miR-505-3p levels remained significantly different between IF and DGF in DCD grafts (P < 0.01), but not in DBD grafts. Receiver operating characteristic curve analysis showed a high sensitivity and specificity (area under the curve, 0.833). CONCLUSIONS: In DCD grafts, high levels of miR-505-3p in preservation fluid are associated with increased risk of DGF after kidney transplantation. Further study is required to confirm the utility of cell-free miR-505-3p as prognostic biomarker for DGF.


Assuntos
MicroRNA Circulante/análise , Função Retardada do Enxerto/etiologia , Transplante de Rim/efeitos adversos , MicroRNAs/análise , Adulto , Idoso , Biomarcadores , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Curva ROC
7.
Stem Cell Reports ; 11(6): 1493-1505, 2018 12 11.
Artigo em Inglês | MEDLINE | ID: mdl-30503260

RESUMO

Predicting developmental potency and risk of posttransplantation tumor formation by human pluripotent stem cells (hPSCs) and their derivatives largely rely on classical histological analysis of teratomas. Here, we investigated whether an assay based on microRNAs (miRNA) in blood plasma is able to detect potentially malignant elements. Several hPSCs and human malignant germ cell tumor (hGCT) lines were investigated in vitro and in vivo after mouse xenografting. The multiple conventional hPSC lines generated mature teratomas, while xenografts from induced hPSCs (hiPSCs) with reactivated reprogramming transgenes and hGCT lines contained undifferentiated and potentially malignant components. The presence of these elements was reflected in the mRNA and miRNA profiles of the xenografts with OCT3/4 mRNA and the miR-371 and miR-302 families readily detectable. miR-371 family members were also identified in mouse plasma faithfully reporting undifferentiated elements in the xenografts. This study demonstrated that undifferentiated and potentially malignant cells could be detected in vivo.


Assuntos
Bioensaio/métodos , Biomarcadores Tumorais/sangue , Diferenciação Celular/genética , MicroRNAs/sangue , Células-Tronco Pluripotentes/metabolismo , Teratoma/sangue , Teratoma/genética , Animais , Biomarcadores Tumorais/genética , Linhagem Celular , Regulação Neoplásica da Expressão Gênica , Humanos , Camundongos , MicroRNAs/genética , Neoplasias Embrionárias de Células Germinativas/genética , Neoplasias Embrionárias de Células Germinativas/patologia , Análise de Componente Principal , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Fatores de Tempo , Ensaios Antitumorais Modelo de Xenoenxerto
8.
PLoS One ; 13(4): e0194259, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29649216

RESUMO

BACKGROUND: Levothyroxine replacement treatment in hypothyroidism is unable to restore physiological thyroxine and triiodothyronine concentrations in serum and tissues completely. Normal serum thyroid stimulating hormone (TSH) concentrations reflect only pituitary euthyroidism and, therefore, novel biomarkers representing tissue-specific thyroid state are needed. MicroRNAs (miRNAs), small non-coding regulatory RNAs, exhibit tissue-specific expression patterns and can be detectable in serum. Previous studies have demonstrated differential expression of (precursors of) miRNAs in tissues under the influence of thyroid hormone. OBJECTIVE: To study if serum miRNA profiles are changed in different thyroid states. DESIGN AND METHODS: We studied 13 athyroid patients (6 males) during TSH suppressive therapy and after 4 weeks of thyroid hormone withdrawal. A magnetic bead capture system was used to isolate 384 defined miRNAs from serum. Subsequently, the TaqMan Array Card 3.0 platform was used for profiling after individual target amplification. RESULTS: Mean age of the subjects was 44.0 years (range 20-61 years). Median TSH levels were 88.9 mU/l during levothyroxine withdrawal and 0.006 mU/l during LT4 treatment with a median dosage of 2.1 µg/kg. After normalization to allow inter-sample analysis, a paired analysis did not demonstrate a significant difference in expression of any of the 384 miRNAs analyzed on and off LT4 treatment. CONCLUSION: Although we previously showed an up-regulation of pri-miRNAs 133b and 206 in hypothyroid state in skeletal muscle, the present study does not supply evidence that thyroid state also affects serum miRNAs in humans.


Assuntos
Hipotireoidismo/tratamento farmacológico , MicroRNAs/sangue , Glândula Tireoide/cirurgia , Hormônios Tireóideos/sangue , Tiroxina/uso terapêutico , Adulto , Biomarcadores/sangue , Feminino , Humanos , Hipotireoidismo/sangue , Masculino , Pessoa de Meia-Idade , Tireotropina/sangue , Tri-Iodotironina/sangue , Adulto Jovem
9.
Medicine (Baltimore) ; 96(30): e7489, 2017 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-28746190

RESUMO

VASA, also known as DDX4, is reported to be specifically expressed in cells belonging to the germ cell lineage, both in males and females. Therefore, it could be an informative protein biomarker to be applied on semen to differentiate between obstructive and nonobstructive azoospermia (OA and NOA, respectively). In addition, it could be of value to predict sperm retrieval based on testicular sperm extraction. Immunocytochemistry of proven OA semen using both polyclonal and monoclonal antibodies against VASA showed positive staining of both cells and cell sized particles. This is spite of being the absolute negative controls, completely lacking germ lineage derived cells and material. In order to identify the source of the VASA-positive material, a detailed screen of different anatomical parts of the whole male urogenital tract was performed of multiple cases using immunohistochemistry.The polyclonal antibody stained, besides the expected germ cells in the testis, epithelium of the bladder and the seminal vesicles. The monoclonal antibody only stained the latter. To investigate whether the immunohistochemical staining is associated with the presence of the corresponding VASA mRNA, samples of seminal vesicles, bladder, testis, and semen (with and without germ cells) were investigated using the specific quantitative reverse transcription-polymerase chain reaction (qRT-PCR) on 42 samples. A positive result was detected in testis and semen containing germ cells (n = 10 and 8), being negative in semen without germ cells (n = 11), bladder (n = 3), and seminal vesicles (n = 10).Two commercially available VASA antibodies (mono- and polyclonal) are not specific. In contrast, VASA-mRNA evaluation, using qRT-PCR, is specific for the presence of germ cells, therefore, is an interesting molecular biomarker for germ cell detection in semen.


Assuntos
RNA Helicases DEAD-box/metabolismo , Células Germinativas , Imuno-Histoquímica , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase em Tempo Real , Anticorpos Monoclonais , Azoospermia/diagnóstico , Azoospermia/metabolismo , Azoospermia/patologia , Biomarcadores/metabolismo , RNA Helicases DEAD-box/imunologia , Células Germinativas/metabolismo , Células Germinativas/patologia , Humanos , Masculino , Sêmen/citologia , Sêmen/metabolismo , Glândulas Seminais/metabolismo , Glândulas Seminais/patologia , Testículo/metabolismo , Testículo/patologia , Fixação de Tecidos , Bexiga Urinária/metabolismo , Bexiga Urinária/patologia
10.
PLoS One ; 12(5): e0178169, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28542371

RESUMO

Adult male germline stem cells (spermatogonia) proliferate by mitosis and, after puberty, generate spermatocytes that undertake meiosis to produce haploid spermatozoa. Germ cells are under evolutionary constraint to curtail mutations and maintain genome integrity. Despite constant turnover, spermatogonia very rarely form tumors, so-called spermatocytic tumors (SpT). In line with the previous identification of FGFR3 and HRAS selfish mutations in a subset of cases, candidate gene screening of 29 SpTs identified an oncogenic NRAS mutation in two cases. To gain insights in the etiology of SpT and into properties of the male germline, we performed whole-genome sequencing of five tumors (4/5 with matched normal tissue). The acquired single nucleotide variant load was extremely low (~0.2 per Mb), with an average of 6 (2-9) non-synonymous variants per tumor, none of which is likely to be oncogenic. The observed mutational signature of SpTs is strikingly similar to that of germline de novo mutations, mostly involving C>T transitions with a significant enrichment in the ACG trinucleotide context. The tumors exhibited extensive aneuploidy (50-99 autosomes/tumor) involving whole-chromosomes, with recurrent gains of chr9 and chr20 and loss of chr7, suggesting that aneuploidy itself represents the initiating oncogenic event. We propose that SpT etiology recapitulates the unique properties of male germ cells; because of evolutionary constraints to maintain low point mutation rate, rare tumorigenic driver events are caused by a combination of gene imbalance mediated via whole-chromosome aneuploidy. Finally, we propose a general framework of male germ cell tumor pathology that accounts for their mutational landscape, timing and cellular origin.


Assuntos
Biomarcadores Tumorais/genética , Genoma Humano , Mutação em Linhagem Germinativa/genética , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Espermatócitos/patologia , Neoplasias Testiculares/genética , Variações do Número de Cópias de DNA/genética , Metilação de DNA , Humanos , Masculino , Receptor Tipo 3 de Fator de Crescimento de Fibroblastos , Maturidade Sexual , Espermatócitos/metabolismo , Neoplasias Testiculares/patologia
11.
Genome Res ; 26(11): 1490-1504, 2016 11.
Artigo em Inglês | MEDLINE | ID: mdl-27803193

RESUMO

Testicular germ cell tumors (TGCTs) share germline ancestry but diverge phenotypically and clinically as seminoma (SE) and nonseminoma (NSE), the latter including the pluripotent embryonal carcinoma (EC) and its differentiated derivatives, teratoma (TE), yolk sac tumor (YST), and choriocarcinoma. Epigenomes from TGCTs may illuminate reprogramming in both normal development and testicular tumorigenesis. Herein we investigate pure-histological forms of 130 TGCTs for conserved and subtype-specific DNA methylation, including analysis of relatedness to pluripotent stem cell (ESC, iPSC), primordial germ cell (PGC), and differentiated somatic references. Most generally, TGCTs conserve PGC-lineage erasure of maternal and paternal genomic imprints and DPPA3 (also known as STELLA); however, like ESCs, TGCTs show focal recurrent imprinted domain hypermethylation. In this setting of shared physiologic erasure, NSEs harbor a malignancy-associated hypermethylation core, akin to that of a diverse cancer compendium. Beyond these concordances, we found subtype epigenetic homology with pluripotent versus differentiated states. ECs demonstrate a striking convergence of both CpG and CpH (non-CpG) methylation with pluripotent states; the pluripotential methyl-CpH signature crosses species boundaries and is distinct from neuronal methyl-CpH. EC differentiation to TE and YST entails reprogramming toward the somatic state, with loss of methyl-CpH but de novo methylation of pluripotency loci such as NANOG Extreme methyl-depletion among SE reflects the PGC methylation nadir. Adjacent to TGCTs, benign testis methylation profiles are determined by spermatogenetic proficiency measured by Johnsen score. In sum, TGCTs share collective entrapment in a PGC-like state of genomic-imprint and DPPA3 erasure, recurrent hypermethylation of cancer-associated targets, and subtype-dependent pluripotent, germline, or somatic methylation.


Assuntos
Reprogramação Celular , Metilação de DNA , Impressão Genômica , Neoplasias Embrionárias de Células Germinativas/genética , Células-Tronco Pluripotentes/metabolismo , Proteínas/genética , Neoplasias Testiculares/genética , Linhagem da Célula , Ilhas de CpG , Regulação Neoplásica da Expressão Gênica , Humanos , Masculino , Proteína Homeobox Nanog/genética , Proteína Homeobox Nanog/metabolismo , Células-Tronco Pluripotentes/citologia , Proteínas/metabolismo
12.
Br J Cancer ; 114(2): 151-62, 2016 Jan 19.
Artigo em Inglês | MEDLINE | ID: mdl-26671749

RESUMO

BACKGROUND: The current biomarkers alpha-fetoprotein and human chorionic gonadotropin have limited sensitivity and specificity for diagnosing malignant germ-cell tumours (GCTs). MicroRNAs (miRNAs) from the miR-371-373 and miR-302/367 clusters are overexpressed in all malignant GCTs, and some of these miRNAs show elevated serum levels at diagnosis. Here, we developed a robust technical pipeline to quantify these miRNAs in the serum and cerebrospinal fluid (CSF). The pipeline was used in samples from a cohort of exclusively paediatric patients with gonadal and extragonadal malignant GCTs, compared with appropriate tumour and non-tumour control groups. METHODS: We developed a method for miRNA quantification that enabled sample adequacy assessment and reliable data normalisation. We performed qRT-PCR profiling for miR-371-373 and miR-302/367 cluster miRNAs in a total of 45 serum and CSF samples, obtained from 25 paediatric patients. RESULTS: The exogenous non-human spike-in cel-miR-39-3p and the endogenous housekeeper miR-30b-5p were optimal for obtaining robust serum and CSF qRT-PCR quantification. A four-serum miRNA panel (miR-371a-3p, miR-372-3p, miR-373-3p and miR-367-3p): (i) showed high sensitivity/specificity for diagnosing paediatric extracranial malignant GCT; (ii) allowed early detection of relapse of a testicular mixed malignant GCT; and (iii) distinguished intracranial malignant GCT from intracranial non-GCT tumours at diagnosis, using CSF and serum samples. CONCLUSIONS: The pipeline we have developed is robust, scalable and transferable. It potentially promises to improve clinical management of paediatric (and adult) malignant GCTs.


Assuntos
Biomarcadores Tumorais/sangue , Neoplasias do Sistema Nervoso Central/diagnóstico , MicroRNAs/sangue , Recidiva Local de Neoplasia/diagnóstico , Neoplasias Embrionárias de Células Germinativas/diagnóstico , Neoplasias Ovarianas/diagnóstico , Neoplasias Testiculares/diagnóstico , Adolescente , Biomarcadores Tumorais/líquido cefalorraquidiano , Carcinoma Embrionário/sangue , Carcinoma Embrionário/líquido cefalorraquidiano , Carcinoma Embrionário/diagnóstico , Neoplasias do Sistema Nervoso Central/sangue , Neoplasias do Sistema Nervoso Central/líquido cefalorraquidiano , Criança , Pré-Escolar , Coriocarcinoma não Gestacional/sangue , Coriocarcinoma não Gestacional/líquido cefalorraquidiano , Coriocarcinoma não Gestacional/diagnóstico , Gonadotropina Coriônica/sangue , Gonadotropina Coriônica/líquido cefalorraquidiano , Tumor do Seio Endodérmico/sangue , Tumor do Seio Endodérmico/líquido cefalorraquidiano , Tumor do Seio Endodérmico/diagnóstico , Feminino , Germinoma/sangue , Germinoma/líquido cefalorraquidiano , Germinoma/diagnóstico , Humanos , Lactente , Recém-Nascido , Masculino , MicroRNAs/líquido cefalorraquidiano , Recidiva Local de Neoplasia/sangue , Recidiva Local de Neoplasia/líquido cefalorraquidiano , Neoplasias Embrionárias de Células Germinativas/sangue , Neoplasias Embrionárias de Células Germinativas/líquido cefalorraquidiano , Neoplasias Ovarianas/sangue , Neoplasias Ovarianas/líquido cefalorraquidiano , Reação em Cadeia da Polimerase , Região Sacrococcígea , Sensibilidade e Especificidade , Neoplasias Testiculares/sangue , Neoplasias Testiculares/líquido cefalorraquidiano , alfa-Fetoproteínas/líquido cefalorraquidiano , alfa-Fetoproteínas/metabolismo
13.
Mol Oncol ; 10(4): 526-37, 2016 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-26654129

RESUMO

Type II germ cell tumors arise after puberty from a germ cell that was incorrectly programmed during fetal life. Failure of testicular germ cells to properly differentiate can lead to the formation of germ cell neoplasia in situ of the testis; this precursor cell invariably gives rise to germ cell cancer after puberty. The Nodal co-receptor Cripto is expressed transiently during normal germ cell development and is ectopically expressed in non-seminomas that arise from germ cell neoplasia in situ, suggesting that its aberrant expression may underlie germ cell dysregulation and hence germ cell cancer. Here we investigated methylation of the Cripto promoter in mouse germ cells and human germ cell cancer and correlated this with the level of CRIPTO protein expression. We found hypomethylation of the CRIPTO promoter in undifferentiated fetal germ cells, embryonal carcinoma and seminomas, but hypermethylation in differentiated fetal germ cells and the differentiated types of non-seminomas. CRIPTO protein was strongly expressed in germ cell neoplasia in situ along with embryonal carcinoma, yolk sac tumor and seminomas. Further, cleaved CRIPTO was detected in media from seminoma and embryonal carcinoma cell lines, suggesting that cleaved CRIPTO may provide diagnostic indication of germ cell cancer. Accordingly, CRIPTO was detectable in serum from 6/15 patients with embryonal carcinoma, 5/15 patients with seminoma, 4/5 patients with germ cell neoplasia in situ cells only and in 1/15 control patients. These findings suggest that CRIPTO expression may be a useful serological marker for diagnostic and/or prognostic purposes during germ cell cancer management.


Assuntos
Carcinoma Embrionário , Fator de Crescimento Epidérmico , Epigênese Genética , Proteínas Ligadas por GPI , Regulação Neoplásica da Expressão Gênica , Peptídeos e Proteínas de Sinalização Intercelular , Glicoproteínas de Membrana , Proteínas de Neoplasias , Neoplasias Testiculares , Animais , Carcinoma Embrionário/sangue , Carcinoma Embrionário/diagnóstico , Carcinoma Embrionário/genética , Fator de Crescimento Epidérmico/biossíntese , Fator de Crescimento Epidérmico/genética , Proteínas Ligadas por GPI/biossíntese , Proteínas Ligadas por GPI/genética , Humanos , Peptídeos e Proteínas de Sinalização Intercelular/biossíntese , Peptídeos e Proteínas de Sinalização Intercelular/genética , Masculino , Glicoproteínas de Membrana/biossíntese , Glicoproteínas de Membrana/genética , Camundongos , Camundongos Transgênicos , Proteínas de Neoplasias/biossíntese , Proteínas de Neoplasias/genética , Neoplasias Testiculares/sangue , Neoplasias Testiculares/diagnóstico , Neoplasias Testiculares/genética
14.
Genome Res ; 25(10): 1536-45, 2015 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-26260970

RESUMO

Somatic L1 retrotransposition events have been shown to occur in epithelial cancers. Here, we attempted to determine how early somatic L1 insertions occurred during the development of gastrointestinal (GI) cancers. Using L1-targeted resequencing (L1-seq), we studied different stages of four colorectal cancers arising from colonic polyps, seven pancreatic carcinomas, as well as seven gastric cancers. Surprisingly, we found somatic L1 insertions not only in all cancer types and metastases but also in colonic adenomas, well-known cancer precursors. Some insertions were also present in low quantities in normal GI tissues, occasionally caught in the act of being clonally fixed in the adjacent tumors. Insertions in adenomas and cancers numbered in the hundreds, and many were present in multiple tumor sections, implying clonal distribution. Our results demonstrate that extensive somatic insertional mutagenesis occurs very early during the development of GI tumors, probably before dysplastic growth.


Assuntos
Neoplasias Gastrointestinais/genética , Elementos Nucleotídeos Longos e Dispersos , Mutagênese Insercional , Progressão da Doença , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Humanos , Proteínas de Neoplasias/biossíntese , Proteínas de Neoplasias/genética , Análise de Sequência com Séries de Oligonucleotídeos , Fatores de Tempo
15.
PLoS One ; 10(4): e0122146, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-25859847

RESUMO

The cell of origin of the five subtypes (I-V) of germ cell tumors (GCTs) are assumed to be germ cells from different maturation stages. This is (potentially) reflected in their methylation status as fetal maturing primordial germ cells are globally demethylated during migration from the yolk sac to the gonad. Imprinted regions are erased in the gonad and later become uniparentally imprinted according to fetal sex. Here, 91 GCTs (type I-IV) and four cell lines were profiled (Illumina's HumanMethylation450BeadChip). Data was pre-processed controlling for cross hybridization, SNPs, detection rate, probe-type bias and batch effects. The annotation was extended, covering snRNAs/microRNAs, repeat elements and imprinted regions. A Hidden Markov Model-based genome segmentation was devised to identify differentially methylated genomic regions. Methylation profiles allowed for separation of clusters of non-seminomas (type II), seminomas/dysgerminomas (type II), spermatocytic seminomas (type III) and teratomas/dermoid cysts (type I/IV). The seminomas, dysgerminomas and spermatocytic seminomas were globally hypomethylated, in line with previous reports and their demethylated precursor. Differential methylation and imprinting status between subtypes reflected their presumed cell of origin. Ovarian type I teratomas and dermoid cysts showed (partial) sex specific uniparental maternal imprinting. The spermatocytic seminomas showed uniparental paternal imprinting while testicular teratomas exhibited partial imprinting erasure. Somatic imprinting in type II GCTs might indicate a cell of origin after global demethylation but before imprinting erasure. This is earlier than previously described, but agrees with the totipotent/embryonic stem cell like potential of type II GCTs and their rare extra-gonadal localization. The results support the common origin of the type I teratomas and show strong similarity between ovarian type I teratomas and dermoid cysts. In conclusion, we identified specific and global methylation differences between GCT subtypes, providing insight into their developmental timing and underlying developmental biology. Data and extended annotation are deposited at GEO (GSE58538 and GPL18809).


Assuntos
Metilação de DNA , Neoplasias Embrionárias de Células Germinativas/genética , Neoplasias Embrionárias de Células Germinativas/patologia , Linhagem Celular Tumoral , Feminino , Genoma Humano , Estudo de Associação Genômica Ampla , Humanos , Masculino , Seminoma/genética , Seminoma/patologia , Teratoma/genética , Teratoma/patologia
16.
PLoS One ; 9(6): e98330, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-24887064

RESUMO

BACKGROUND: Originating from Primordial Germ Cells/gonocytes and developing via a precursor lesion called Carcinoma In Situ (CIS), Germ Cell Cancers (GCC) are the most common cancer in young men, subdivided in seminoma (SE) and non-seminoma (NS). During physiological germ cell formation/maturation, epigenetic processes guard homeostasis by regulating the accessibility of the DNA to facilitate transcription. Epigenetic deregulation through genetic and environmental parameters (i.e. genvironment) could disrupt embryonic germ cell development, resulting in delayed or blocked maturation. This potentially facilitates the formation of CIS and progression to invasive GCC. Therefore, determining the epigenetic and functional genomic landscape in GCC cell lines could provide insight into the pathophysiology and etiology of GCC and provide guidance for targeted functional experiments. RESULTS: This study aims at identifying epigenetic footprints in SE and EC cell lines in genome-wide profiles by studying the interaction between gene expression, DNA CpG methylation and histone modifications, and their function in the pathophysiology and etiology of GCC. Two well characterized GCC-derived cell lines were compared, one representative for SE (TCam-2) and the other for EC (NCCIT). Data were acquired using the Illumina HumanHT-12-v4 (gene expression) and HumanMethylation450 BeadChip (methylation) microarrays as well as ChIP-sequencing (activating histone modifications (H3K4me3, H3K27ac)). Results indicate known germ cell markers not only to be differentiating between SE and NS at the expression level, but also in the epigenetic landscape. CONCLUSION: The overall similarity between TCam-2/NCCIT support an erased embryonic germ cell arrested in early gonadal development as common cell of origin although the exact developmental stage from which the tumor cells are derived might differ. Indeed, subtle difference in the (integrated) epigenetic and expression profiles indicate TCam-2 to exhibit a more germ cell-like profile, whereas NCCIT shows a more pluripotent phenotype. The results provide insight into the functional genome in GCC cell lines.


Assuntos
Carcinoma Embrionário/genética , Epigênese Genética , Perfilação da Expressão Gênica , Neoplasias Embrionárias de Células Germinativas/genética , Seminoma/genética , Carcinoma Embrionário/patologia , Linhagem Celular Tumoral , Metilação de DNA , Humanos , Neoplasias Embrionárias de Células Germinativas/patologia , Seminoma/patologia
17.
PLoS One ; 9(1): e83585, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-24404135

RESUMO

The transcription factor SOX2, associated with amongst others OCT3/4, is essential for maintenance of pluripotency and self-renewal of embryonic stem cells. SOX2 is highly expressed in embryonal carcinoma (EC), the stem cell component of malignant nonseminomatous germ cell tumors, referred to as germ cell cancer (GCC). In fact, OCT3/4 together with SOX2 is an informative diagnostic tool for EC in a clinical setting. Several studies support the hypothesis that SOX2 is a relevant oncogenic factor in various cancers and recently, SOX2 has been suggested as a putative therapeutic target for early stage EC. We demonstrate the presence of genomic amplification of SOX2 in an EC cell line, NCCIT, using array comparative genome hybridization and fluorescence in situ hybridization. Down-regulation of SOX2 by targeted siRNA provokes NCCIT cells towards apoptosis, while inhibition of OCT3/4 expression induced differentiation, with retained SOX2 levels. Mice pluripotent xenografts from NCCIT (N-NCCIT and N2-NCCIT) show a consistent SOX2 expression, in spite of loss of the expression of OCT3/4, and differentiation, with retained presence of genomic amplification. No SOX2 amplification has been identified in primary pure and mixed EC in vivo patient samples so far. The data presented in this study are based on a single EC cell line with a SOX2 amplification, with NT2 as control EC cell line, showing no profound induction of apoptosis upon SOX2 downregulation. The findings are of relevance to identify mechanisms involved in the pathogenesis of EC tumors, and support the model of SOX2-oncogene dependency of EC, which however, does not exclude induction of differentiation. This finding is likely related to the presence of wild type p53 in GCC, resulting in expression of downstream target genes, amongst others miR-34a, miR-145 and SOX2, associated to the unique sensitivity of GCC to DNA damaging agents.


Assuntos
Carcinoma Embrionário/genética , Carcinoma Embrionário/metabolismo , Fatores de Transcrição SOXB1/genética , Fatores de Transcrição SOXB1/metabolismo , Animais , Apoptose/genética , Biomarcadores Tumorais , Linhagem Celular Tumoral , Modelos Animais de Doenças , Feminino , Amplificação de Genes , Expressão Gênica , Técnicas de Silenciamento de Genes , Inativação Gênica , Xenoenxertos , Humanos , Camundongos , Modelos Biológicos , Fator 3 de Transcrição de Octâmero/genética , Fator 3 de Transcrição de Octâmero/metabolismo , Interferência de RNA
18.
J Proteomics ; 96: 300-13, 2014 Jan 16.
Artigo em Inglês | MEDLINE | ID: mdl-24269351

RESUMO

UNLABELLED: We analysed the effects of all-trans retinoic acid (ATRA) on proliferation and changes in the global proteome of the nullipotent human embryonal carcinoma cell line 2102Ep and the pluripotent cell line NTERA2 cl.D1 (NT2). Differentially expressed proteins were assessed by 2D-PAGE and mass spectrometry, followed by verification and analysis of protein modifications of proteins of the retinoid pathway. We established a proteome map of the germ cell tumor (GCT) cell line NT2 showing neuronal differentiation under ATRA treatment for 7days. Using bioinformatic analyses, we identified functional groups of altered proteins and potentially involved pathways, of which changes to the organization of the cytoskeleton and anti-apoptotic effects were the most prominent. Changes observed in the expression of factors involved in the retinoid pathway under ATRA, namely an upregulation of CRBP and CRABP2, were also reflected in GCT tissues of different histologies, providing further insight into factors involved in the differentiation of these pluripotent tumors. BIOLOGICAL SIGNIFICANCE: Treatment of NT2 germ cell tumor cells with all-trans retinoic acid (ATRA) is a model to investigate differentiation. We analysed differentially expressed proteins by 2D-PAGE and mass spectrometry and provide a proteome map of NT2 cells under 7days of ATRA. By bioinformatic analyses, functional groups of proteins and involved pathways like changes to the cytoskeleton and anti-apoptotic effects were identified. Factors involved in the retinoid pathway, in particular upregulation of CRBP, CRABP1 and CRABP2, also showed differential expression in tumors with different histological subtypes, which provides insight into gene regulation under induced and spontaneous differentiation in germ cell tumors.


Assuntos
Antineoplásicos/farmacologia , Carcinoma Embrionário/metabolismo , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Proteínas de Neoplasias/biossíntese , Processamento de Proteína Pós-Traducional/efeitos dos fármacos , Proteoma/biossíntese , Tretinoína/farmacologia , Carcinoma Embrionário/patologia , Diferenciação Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Humanos , Fatores de Tempo , Regulação para Cima/efeitos dos fármacos
19.
Mol Oncol ; 7(6): 1083-92, 2013 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-24012110

RESUMO

Germ cell cancers (GCC) are the most frequent malignancy in young Caucasian males. GCC can consist of seminomas (SE) and non-seminomas (malignant NS: embryonal carcinoma (EC), yolk sac tumor (YS), choriocarcinoma (CH) and teratoma (TE)). Current serum-markers used for diagnosis and follow-up (AFP, hCG) are predominantly related to YS and CH and marker positivity can vary during disease. Therefore, stable markers consistently identifying more GCC components, specifically the stem cell components SE and EC, are of interest. Expression of the embryonic stem cell miR-371-3 and miR-302/367 clusters in SE/EC/YS suggest possible application of these micro-RNAs as GCC tumor-markers. The TSmiR protocol constitutes a complete, quality-controlled pipeline for the detection of miRs in serum, based on magnetic bead-based purification and qPCR quantification. As a proof of principle, TSmiR was applied to five independent serum sample series including 80 GCCs, 47 controls, 11 matched pre/post orchidectomy samples and 12 no-GCC testicular masses. GCC serum samples showed a consistent, significant (p < 0.0064) increase of miR-371/372/373/367 levels. Analogous, serum levels returned to baseline after orchidectomy (stage-I disease). Moreover, there was a trend toward higher miR levels in patients with metastasis. These results imply suitability for diagnosis and follow-up. TSmiR showed an overall sensitivity of 98%, clearly outperforming the traditional serum markers AFP/hCG (36%/57%, sensitivity(AFP) = 3%/45%; sensitivity(hCG) = 62%/66%, SE/NS). TSmiR misclassified one tumor as a control. Serum AFP/hCG and TSmiR combined identified all T samples correctly. In conclusion, TSmiR constitutes a highly sensitive and reproducible serum test for GCC patients, suitable to be prospectively tested for diagnostic and follow-up purposes.


Assuntos
Biomarcadores Tumorais/metabolismo , MicroRNAs/metabolismo , Neoplasias Embrionárias de Células Germinativas/diagnóstico , Neoplasias Embrionárias de Células Germinativas/metabolismo , RNA Neoplásico/metabolismo , Neoplasias Testiculares/diagnóstico , Neoplasias Testiculares/metabolismo , Adolescente , Adulto , Biomarcadores Tumorais/genética , Criança , Pré-Escolar , Humanos , Masculino , MicroRNAs/genética , Neoplasias Embrionárias de Células Germinativas/genética , Neoplasias Embrionárias de Células Germinativas/patologia , Células-Tronco Neoplásicas/metabolismo , Células-Tronco Neoplásicas/patologia , RNA Neoplásico/genética , Neoplasias Testiculares/genética , Neoplasias Testiculares/patologia
20.
Bioinformatics ; 29(13): 1638-46, 2013 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-23640718

RESUMO

MOTIVATION: Algorithms predicting microRNA (miR)-mRNA interactions generate high numbers of possible interactions, many of which might be non-existent or irrelevant in a certain biological context. It is desirable to develop a transparent, user-friendly, unbiased tool to enrich miR-mRNA predictions. RESULTS: The miMsg algorithm uses matched miR/mRNA expression data to enrich miR-mRNA predictions. It grades interactions by the number, magnitude and significance of misplacements in the combined ranking profiles of miR/mRNA expression assessed over multiple biological samples. miMsg requires minimal user input and makes no statistical assumptions. It identified 921 out of 56 262 interactions as top scoring and significant in an actual germ cell cancer dataset. Twenty-eight miR-mRNA pairs were deemed of highest interest based on ranking by miMsg and supported by current knowledge about validated interactions and biological function. To conclude, miMsg is an effective algorithm to reduce a high number of predicted interactions to a small set of high confidence interactions for further study. AVAILABILITY AND IMPLEMENTATION: Matlab source code and datasets available at www.martinrijlaarsdam.nl/mimsg . SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.


Assuntos
Algoritmos , MicroRNAs/metabolismo , RNA Mensageiro/metabolismo , Perfilação da Expressão Gênica , Humanos , Neoplasias Embrionárias de Células Germinativas/genética , Neoplasias Embrionárias de Células Germinativas/metabolismo
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA