RESUMEN
PURPOSE: Current clinical guidelines recommend cystoscopy in patients who present with hematuria to rule out a bladder tumor. We evaluated whether our previously developed urine assay was able to triage patients with hematuria for cystoscopy in a large prospective cohort. MATERIALS AND METHODS: A urine sample was collected before cystoscopy and mutation/methylation status of 6 genes was determined on cellular DNA. The existing diagnostic model was validated on this cohort. Logistic regression was applied to investigate other potential variables. The primary end point was the model performance as indicated by the AUC. Secondary end points were sensitivity, specificity and negative predictive value. Clinical usefulness was determined by the net benefit approach. RESULTS: In 838 patients biomarker status could be determined for all genes. Urothelial cancer was observed in 112 patients (98 of 457 in the gross and 14 of 381 in the microscopic hematuria group). Validation of the existing model resulted in an AUC of 0.93. Logistic regression analysis identified type of hematuria as a significant additional variable. Adding type of hematuria resulted in an AUC of 0.95 (96% sensitivity, 73% specificity, 99% negative predictive value). The assay identified all upper tract tumors not visible by cystoscopy (in 6). Net benefit analysis showed that the urine assay should be preferred over current clinical practice. Implementing the urine assay as a triage tool could lead to a 53% reduction in cystoscopies. CONCLUSIONS: The urine assay detected urothelial cancer with a very high accuracy and can be used to triage patients presenting with hematuria for cystoscopy.
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Biomarcadores de Tumor , Metilación de ADN , Análisis Mutacional de ADN , Hematuria , Neoplasias de la Vejiga Urinaria/diagnóstico , Neoplasias de la Vejiga Urinaria/genética , Adulto , Anciano , Anciano de 80 o más Años , Biomarcadores de Tumor/genética , Biomarcadores de Tumor/orina , Estudios de Cohortes , Cistoscopía , Femenino , Hematuria/genética , Hematuria/orina , Proteínas de Homeodominio/genética , Humanos , Masculino , Persona de Mediana Edad , Países Bajos , Factores de Transcripción Otx/genética , Valor Predictivo de las Pruebas , Proteínas Proto-Oncogénicas p21(ras)/genética , Receptor Tipo 3 de Factor de Crecimiento de Fibroblastos/genética , Sensibilidad y Especificidad , Telomerasa/genética , Factores de Transcripción/genética , Triaje , Neoplasias de la Vejiga Urinaria/orina , Adulto JovenRESUMEN
BACKGROUND: The septin 9 gene (SEPT9) codes for a GTP-binding protein associated with filamentous structures and cytoskeleton formation. SEPT9 plays a role in multiple cancers as either an oncogene or a tumor suppressor gene. Regulation of SEPT9 expression is complex and not well understood; however, hypermethylation of the gene was recently introduced as a biomarker for early detection of colorectal cancer (CRC) and has been linked to cancer of the breast and of the head and neck. Because the DNA methylation landscape of different regions of SEPT9 is poorly understood in cancer, we analyzed the methylation patterns of this gene in distinct cell populations from normal and diseased colon mucosa. METHODS: Laser capture microdissection was performed to obtain homogeneous populations of epithelial and stromal cells from normal, adenomatous, and tumorous colon mucosa. Microdissected samples were analyzed using direct bisulfite sequencing to determine the DNA methylation status of eight regions within and near the SEPT9 gene. Septin-9 protein expression was assessed using immunohistochemistry (IHC). RESULTS: Regions analyzed in SEPT9 were unmethylated in normal tissue except for a methylation boundary detected downstream of the largest CpG island. In adenoma and tumor tissues, epithelial cells displayed markedly increased DNA methylation levels (>80%, p <0.0001) in only one of the CpG islands investigated. SEPT9 methylation in stromal cells increased in adenomatous and tumor tissues (≤50%, p <0.0001); however, methylation did not increase in stromal cells of normal tissue close to the tumor. IHC data indicated a significant decrease (p <0.01) in Septin-9 protein levels in epithelial cells derived from adenoma and tumor tissues; Septin-9 protein levels in stromal cells were low in all tissues. CONCLUSIONS: Hypermethylation of SEPT9 in adenoma and CRC specimens is confined to one of several CpG islands of this gene. Tumor-associated aberrant methylation originates in epithelial cells; stromal cells appear to acquire hypermethylation subsequent to epithelial cells, possibly through field effects. The region in SEPT9 with disease-related hypermethylation also contains the CpGs targeted by a novel blood-based screening test (Epi proColon®), providing further support for the clinical relevance of this biomarker.
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Neoplasias Colorrectales/genética , Islas de CpG , Metilación de ADN , Septinas/genética , Adulto , Anciano , Neoplasias Colorrectales/patología , Femenino , Orden Génico , Humanos , Inmunohistoquímica , Masculino , Persona de Mediana Edad , Clasificación del Tumor , Estadificación de Neoplasias , Septinas/metabolismo , Adulto JovenRESUMEN
BACKGROUND: Various biomarkers for prediction of distant metastasis in lymph-node negative breast cancer have been described; however, predictive biomarkers for patients with lymph-node positive (LNP) disease in the context of distinct systemic therapies are still very much needed. DNA methylation is aberrant in breast cancer and is likely to play a major role in disease progression. In this study, the DNA methylation status of 202 candidate loci was screened to identify those loci that may predict outcome in LNP/estrogen receptor-positive (ER+) breast cancer patients with adjuvant anthracycline-based chemotherapy. METHODS: Quantitative bisulfite sequencing was used to analyze DNA methylation biomarker candidates in a retrospective cohort of 162 LNP/ER+ breast cancer patients, who received adjuvant anthracycline-based chemotherapy. First, twelve breast cancer specimens were analyzed for all 202 candidate loci to exclude genes that showed no differential methylation. To identify genes that predict distant metastasis, the remaining loci were analyzed in 84 selected cases, including the 12 initial ones. Significant loci were analyzed in the remaining 78 independent cases. Metastasis-free survival analysis was conducted by using Cox regression, time-dependent ROC analysis, and the Kaplan-Meier method. Pairwise multivariate regression analysis was performed by linear Cox Proportional Hazard models, testing the association between methylation scores and clinical parameters with respect to metastasis-free survival. RESULTS: Of the 202 loci analysed, 37 showed some indication of differential DNA methylation among the initial 12 patient samples tested. Of those, 6 loci were associated with outcome in the initial cohort (n = 84, log rank test, p < 0.05).Promoter DNA methylation of cysteine dioxygenase 1 (CDO1) was confirmed in univariate and in pairwise multivariate analysis adjusting for age at surgery, pathological T stage, progesterone receptor status, grade, and endocrine therapy as a strong and independent biomarker for outcome prediction in the independent validation set (log rank test p-value = 0.0010). CONCLUSIONS: CDO1 methylation was shown to be a strong predictor for distant metastasis in retrospective cohorts of LNP/ER+ breast cancer patients, who had received adjuvant anthracycline-based chemotherapy.
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Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Biomarcadores de Tumor , Neoplasias de la Mama/tratamiento farmacológico , Neoplasias de la Mama/genética , Cisteína-Dioxigenasa/genética , Metilación de ADN , Regiones Promotoras Genéticas , Receptores de Estrógenos/análisis , Adulto , Anciano , Anciano de 80 o más Años , Antraciclinas/administración & dosificación , Antibióticos Antineoplásicos/administración & dosificación , Biomarcadores de Tumor/análisis , Biomarcadores de Tumor/genética , Neoplasias de la Mama/enzimología , Neoplasias de la Mama/mortalidad , Neoplasias de la Mama/secundario , Quimioterapia Adyuvante , Europa (Continente) , Femenino , Humanos , Estimación de Kaplan-Meier , Metástasis Linfática , Persona de Mediana Edad , Estadificación de Neoplasias , Valor Predictivo de las Pruebas , Modelos de Riesgos Proporcionales , Curva ROC , Estudios Retrospectivos , Medición de Riesgo , Factores de Riesgo , Factores de Tiempo , Resultado del TratamientoRESUMEN
A procedure for simultaneous quantification of DNA methylation of several genes in minute amounts of sample material was developed and applied to microdissected formalin-fixed and paraffin-embedded breast tissues. The procedure is comprised of an optimized bisulfite treatment protocol suitable for samples containing only few cells, a multiplex preamplification and subsequent locus specific reamplification, and a novel quantitative bisulfite sequencing method based on the incorporation of a normalization domain into the PCR product. A real-time PCR assay amplifying repetitive elements was established to quantify low amounts of bisulfite-treated DNA. Ten prognostic and diagnostic epigenetic breast cancer biomarkers (PITX2, RASSF1A, PLAU, LHX3, PITX3, LIMK1, SLITRK1, SLIT2, HS3ST2, and TFF1) were analyzed in tissue samples obtained from two patients with invasive ductal carcinoma of the breast. The microdissected samples were obtained from several areas within the tumor tissue, including intraductal and invasive carcinoma, adenosis, and normal ductal epithelia of adjacent normal tissue, as well as stroma, tumor infiltrating lymphocytes, and adipose tissue. Overall, reliable quantification was possible for all genes. For most genes, increased DNA methylation in invasive and intraductal carcinoma cells compared with other tissue components was observed. For TFF1, decreased methylation levels were observed in tumor cells.
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Biomarcadores de Tumor/análisis , Neoplasias de la Mama/química , Metilación de ADN , Fijadores , Formaldehído , Adhesión en Parafina , Tejido Adiposo/química , Tejido Adiposo/patología , Biomarcadores de Tumor/genética , Neoplasias de la Mama/patología , Carcinoma Ductal de Mama/química , Carcinoma Ductal de Mama/patología , Carcinoma Intraductal no Infiltrante/química , Carcinoma Intraductal no Infiltrante/patología , Femenino , Enfermedad Fibroquística de la Mama/química , Enfermedad Fibroquística de la Mama/patología , Humanos , Indicadores y Reactivos , Rayos Láser , Linfocitos Infiltrantes de Tumor/química , Microdisección , Reacción en Cadena de la Polimerasa/métodos , Regiones Promotoras Genéticas , Análisis de Secuencia de ADN/métodos , Células del Estroma/química , SulfitosRESUMEN
A substantial proportion of human genes contain tissue-specifically DNA-methylated regions (TDMRs). However, little is known about the evolutionary conservation of differentially methylated loci, how they evolve, and the signals that regulate them. We have studied TDMR conservation in the PLG and TBX gene families and in 32 pseudogene-parental gene pairs. Among the members of the recently evolved PLG gene family, 5'-UTR methylation is conserved and inversely correlated with the cognate gene expression, indicating as well a conserved regulatory role of DNA methylation. Conversely, many genes of the much older TBX family display complementary tissue-specific methylation, suggesting an epigenetic complementation in the evolution of this gene family. Similar to gene families, unprocessed pseudogenes arose from gene duplications and we found TDMR conservation in some pseudogene-parental gene pairs displaying short evolutionary distances. However, for the majority of unprocessed pseudogenes and for all processed pseudogenes examined, we found that tissue-specific methylation arose de novo after gene duplication.
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Metilación de ADN , Evolución Molecular , Plasminógeno/genética , Seudogenes , Proteínas de Dominio T Box/genética , Regiones no Traducidas 5'/metabolismo , Animales , Secuencia de Bases , Secuencia Conservada , Duplicación de Gen , Humanos , Datos de Secuencia Molecular , Distribución TisularRESUMEN
OBJECTIVE: To evaluate an epigenetic assay performed on tissue from negative prostate biopsies in a group of African American (AA) men undergoing repeat biopsy, and to compare accuracy for predicting repeat biopsy outcome to prior studies conducted in predominantly Caucasian populations. MATERIALS AND METHODS: The study population consisted of 211 AA men from 7 urology centers across the United States; all of whom were undergoing 12-core transrectal ultrasound-guided repeat biopsy within 30 months from a negative index biopsy. All biopsy cores from the negative index biopsy were profiled for the epigenetic biomarkers GSTP1, APC, and RASSF1 using ConfirmMDx for Prostate Cancer (MDxHealth, Irvine, CA). RESULTS: Upon repeat biopsy, 130 of 211 subjects (62%) had no prostate cancer (PCa) detected and 81 of 211 (38%) were diagnosed with PCa. Of the subjects with PCa, 54 (67%) were diagnosed with Gleason score (GS) ≤6 PCa and 27 (33%) with GS ≥7 disease. For detection of PCa at repeat biopsy, ConfirmMDx sensitivity was 74.1% and specificity was 60.0%, equivalent to prior studies (P = .235 and .697, respectively). For detection of GS ≥7 PCa, sensitivity was 78% and specificity was 53%. The negative predictive values for detection of all PCa and GS ≥7 PCa were 78.8% and 94.2%, respectively. CONCLUSION: In this group of AA men, we successfully validated an epigenetic assay to assess the need for repeat biopsy. Results were consistent with previous studies from predominantly Caucasian populations. Therefore, the ConfirmMDx assay is a useful tool for risk stratification of AA men who had an initial negative biopsy.
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Biomarcadores de Tumor/genética , Negro o Afroamericano , Epigénesis Genética , Biopsia Guiada por Imagen/métodos , Próstata/patología , Neoplasias de la Próstata/diagnóstico , Adulto , Anciano , Anciano de 80 o más Años , Femenino , Humanos , Masculino , Persona de Mediana Edad , Clasificación del Tumor , Pronóstico , Neoplasias de la Próstata/etnología , Neoplasias de la Próstata/genética , Reproducibilidad de los Resultados , Estudios Retrospectivos , Estados Unidos/epidemiologíaRESUMEN
Preeclampsia is a disease of the mother, fetus, and placenta, and the gaps in our understanding of the complex interactions among their respective disease pathways preclude successful treatment and prevention. The placenta has a key role in the pathogenesis of the terminal pathway characterized by exaggerated maternal systemic inflammation, generalized endothelial damage, hypertension, and proteinuria. This sine qua non of preeclampsia may be triggered by distinct underlying mechanisms that occur at early stages of pregnancy and induce different phenotypes. To gain insights into these molecular pathways, we employed a systems biology approach and integrated different "omics," clinical, placental, and functional data from patients with distinct phenotypes of preeclampsia. First trimester maternal blood proteomics uncovered an altered abundance of proteins of the renin-angiotensin and immune systems, complement, and coagulation cascades in patients with term or preterm preeclampsia. Moreover, first trimester maternal blood from preterm preeclamptic patients in vitro dysregulated trophoblastic gene expression. Placental transcriptomics of women with preterm preeclampsia identified distinct gene modules associated with maternal or fetal disease. Placental "virtual" liquid biopsy showed that the dysregulation of these disease gene modules originates during the first trimester. In vitro experiments on hub transcription factors of these gene modules demonstrated that DNA hypermethylation in the regulatory region of ZNF554 leads to gene down-regulation and impaired trophoblast invasion, while BCL6 and ARNT2 up-regulation sensitizes the trophoblast to ischemia, hallmarks of preterm preeclampsia. In summary, our data suggest that there are distinct maternal and placental disease pathways, and their interaction influences the clinical presentation of preeclampsia. The activation of maternal disease pathways can be detected in all phenotypes of preeclampsia earlier and upstream of placental dysfunction, not only downstream as described before, and distinct placental disease pathways are superimposed on these maternal pathways. This is a paradigm shift, which, in agreement with epidemiological studies, warrants for the central pathologic role of preexisting maternal diseases or perturbed maternal-fetal-placental immune interactions in preeclampsia. The description of these novel pathways in the "molecular phase" of preeclampsia and the identification of their hub molecules may enable timely molecular characterization of patients with distinct preeclampsia phenotypes.
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Enfermedades Placentarias , Preeclampsia , Adulto , Biomarcadores/sangre , Femenino , Humanos , Enfermedades Placentarias/sangre , Enfermedades Placentarias/genética , Enfermedades Placentarias/fisiopatología , Preeclampsia/sangre , Preeclampsia/genética , Preeclampsia/fisiopatología , Embarazo , Proteómica , Biología de Sistemas , Trofoblastos/metabolismo , Trofoblastos/patologíaRESUMEN
Cellular differentiation involves widespread epigenetic reprogramming, including modulation of DNA methylation patterns. Using Differential Methylation Hybridization (DMH) in combination with a custom DMH array containing 51,243 features covering more than 16,000 murine genes, we carried out a genome-wide screen for cell- and tissue-specific differentially methylated regions (tDMRs) in undifferentiated embryonic stem cells (ESCs), in in-vitro induced neural stem cells (NSCs) and 8 differentiated embryonic and adult tissues. Unsupervised clustering of the generated data showed distinct cell- and tissue-specific DNA methylation profiles, revealing 202 significant tDMRs (p<0.005) between ESCs and NSCs and a further 380 tDMRs (p<0.05) between NSCs/ESCs and embryonic brain tissue. We validated these tDMRs using direct bisulfite sequencing (DBS) and methylated DNA immunoprecipitation on chip (MeDIP-chip). Gene ontology (GO) analysis of the genes associated with these tDMRs showed significant (absolute Z score>1.96) enrichment for genes involved in neural differentiation, including, for example, Jag1 and Tcf4. Our results provide robust evidence for the relevance of DNA methylation in early neural development and identify novel marker candidates for neural cell differentiation.