A Novel Epigenetic Signature for Early Diagnosis in Lung Cancer.

PURPOSE: Lung cancer remains as the leading cause of cancer-related death worldwide, mainly due to late diagnosis. Cytology is the gold-standard method for lung cancer diagnosis in minimally invasive respiratory samples, despite its low sensitivity. We aimed to identify epigenetic biomarkers with clinical utility for cancer diagnosis in minimally/noninvasive specimens to improve accuracy of current technologies. EXPERIMENTAL DESIGN: The identification of novel epigenetic biomarkers in stage I lung tumors was accomplished using an integrative genome-wide restrictive analysis of two different large public databases. DNA methylation levels for the selected biomarkers were validated by pyrosequencing in paraffin-embedded tissues and minimally invasive and noninvasive respiratory samples in independent cohorts. RESULTS: We identified nine cancer-specific hypermethylated genes in early-stage lung primary tumors. Four of these genes presented consistent CpG island hypermethylation compared with nonmalignant lung and were associated with transcriptional silencing. A diagnostic signature was built using multivariate logistic regression model based on the combination of four genes: BCAT1, CDO1, TRIM58, and ZNF177 Clinical diagnostic value was also validated in multiple independent cohorts and yielded a remarkable diagnostic accuracy in all cohorts tested. Calibrated and cross-validated epigenetic model predicts with high accuracy the probability to detect cancer in minimally and noninvasive samples. We demonstrated that this epigenetic signature achieved higher diagnostic efficacy in bronchial fluids as compared with conventional cytology for lung cancer diagnosis. CONCLUSIONS: Minimally invasive epigenetic biomarkers have emerged as promising tools for cancer diagnosis. The herein obtained epigenetic model in combination with current diagnostic protocols may improve early diagnosis and outcome of lung cancer patients. Clin Cancer Res; 22(13); 3361-71. ©2016 AACR.

DNA methylation-based prognosis and epidrivers in hepatocellular carcinoma.

UNLABELLED: Epigenetic deregulation has emerged as a driver in human malignancies. There is no clear understanding of the epigenetic alterations in hepatocellular carcinoma (HCC) and of the potential role of DNA methylation markers as prognostic biomarkers. Analysis of tumor tissue from 304 patients with HCC treated with surgical resection allowed us to generate a methylation-based prognostic signature using a training-validation scheme. Methylome profiling was done with the Illumina HumanMethylation450 array (Illumina, Inc., San Diego, CA), which covers 96% of known cytosine-phosphate-guanine (CpG) islands and 485,000 CpG, and transcriptome profiling was performed with Affymetrix Human Genome U219 Plate (Affymetrix, Inc., Santa Clara, CA) and miRNA Chip 2.0. Random survival forests enabled us to generate a methylation signature based on 36 methylation probes. We computed a risk score of mortality for each individual that accurately discriminated patient survival both in the training (221 patients; 47% hepatitis C-related HCC) and validation sets (n = 83; 47% alcohol-related HCC). This signature correlated with known predictors of poor outcome and retained independent prognostic capacity of survival along with multinodularity and platelet count. The subset of patients identified by this signature was enriched in the molecular subclass of proliferation with progenitor cell features. The study confirmed a high prevalence of genes known to be deregulated by aberrant methylation in HCC (e.g., Ras association [RalGDS/AF-6] domain family member 1, insulin-like growth factor 2, and adenomatous polyposis coli) and other solid tumors (e.g., NOTCH3) and describes potential candidate epidrivers (e.g., septin 9 and ephrin B2). CONCLUSIONS: A validated signature of 36 DNA methylation markers accurately predicts poor survival in patients with HCC. Patients with this methylation profile harbor messenger RNA-based signatures indicating tumors with progenitor cell features.

Molecular analysis of chylomicronemia in a clinical laboratory setting: diagnosis of 13 cases of lipoprotein lipase deficiency.

BACKGROUND: Familial chylomicronemia (type I hyperlipidemia) is a rare autosomal recessive disease due mainly to rare variants in the lipoprotein lipase (LPL) gene sequence. Molecular diagnosis of LPL deficiency is now a requirement for the first gene therapy treatment approved in the European Union. Altered coding sequence variants in APOC2, APOA5 or GPIHBP-1 can also cause familial chylomicronemia. Herein, we report the results of our molecular diagnostic activity in this topic, carried out in the setting of a Spanish clinical practice hospital laboratory, which was also extended to some patients who were more likely to have type V hyperlipidemia. METHODS: Samples from twenty-nine unrelated probands with severe hypertriglyceridemia were referred for molecular diagnosis. Samples were first screened for LPL sequence variants by DNA sequencing and, in the absence of alterations, subsequent analysis of APOC2, APOA5, and GPIHBP1 genes was undertaken. Analysis of LPL function in vitro was further studied in two previously uncharacterized LPL sequence variants. RESULTS: Fourteen different, loss-of-function variants were found in the LPL gene: 4 were novel or uncharacterized allelic variants, and of these, 2 were directly shown to affect function. Twenty of 29 probands presented at least one LPL gene allele variant: 8 were homozygous, 9 compound heterozygous and 3 heterozygous. In 13 probands, the finding of two loss-of-function variants supported the diagnosis of LPL deficiency. None of the probands presented sequence variants in the APOC2 gene, whereas 3 presented rare variants within the APOA5 gene. Four of the five patients heterozygous for a common variant in the GPIHBP-1 gene also carried APOA5 sequence variants. CONCLUSIONS: Loss-of-function LPL variants leading to familial chylomicronemia were found in 13 patients, accounting for a significant proportion of the LPL-deficient patients predicted to live in Spain.

A prognostic DNA methylation signature for stage I non-small-cell lung cancer.

PURPOSE: Non-small-cell lung cancer (NSCLC) is a tumor in which only small improvements in clinical outcome have been achieved. The issue is critical for stage I patients for whom there are no available biomarkers that indicate which high-risk patients should receive adjuvant chemotherapy. We aimed to find DNA methylation markers that could be helpful in this regard. PATIENTS AND METHODS: A DNA methylation microarray that analyzes 450,000 CpG sites was used to study tumoral DNA obtained from 444 patients with NSCLC that included 237 stage I tumors. The prognostic DNA methylation markers were validated by a single-methylation pyrosequencing assay in an independent cohort of 143 patients with stage I NSCLC. RESULTS: Unsupervised clustering of the 10,000 most variable DNA methylation sites in the discovery cohort identified patients with high-risk stage I NSCLC who had shorter relapse-free survival (RFS; hazard ratio [HR], 2.35; 95% CI, 1.29 to 4.28; P = .004). The study in the validation cohort of the significant methylated sites from the discovery cohort found that hypermethylation of five genes was significantly associated with shorter RFS in stage I NSCLC: HIST1H4F, PCDHGB6, NPBWR1, ALX1, and HOXA9. A signature based on the number of hypermethylated events distinguished patients with high- and low-risk stage I NSCLC (HR, 3.24; 95% CI, 1.61 to 6.54; P = .001). CONCLUSION: The DNA methylation signature of NSCLC affects the outcome of stage I patients, and it can be practically determined by user-friendly polymerase chain reaction assays. The analysis of the best DNA methylation biomarkers improved prognostic accuracy beyond standard staging.

Epigenetic profiling joins personalized cancer medicine.

Personalized medicine is defined by therapy decisions tailored to individual patients, aiming to improve therapeutic efficiencies and to minimize side effects. The current clinical practice includes targeted therapies for disease-related alterations and molecular biomarker-based patient stratification. However, recent advances in screening technologies have enabled more comprehensive identification strategies and suggest a plethora of additional valuable biomarkers and druggable molecules for future clinical applications. Beside genetic alterations, in particular, DNA methylation biomarkers emerge into the field by presenting stable DNA modifications with predictive potential for drug treatment efficiencies, especially in a cancer context. Although not directly affecting the genetic code, DNA methylation exhibits regulatory functions with high impact on disease onset and progression. In this article, the authors summarize the current knowledge of DNA methylation biomarkers for treatment efficiencies and evaluate their translational value into clinical use.

The cholesterol content of Western diets plays a major role in the paradoxical increase in high-density lipoprotein cholesterol and upregulates the macrophage reverse cholesterol transport pathway.

OBJECTIVE: A high-saturated fatty acid- and cholesterol-containing (HFHC) diet is considered to be a major risk factor for cardiovascular disease. The present study aimed to determine the effects of this Western-type diet on high-density lipoprotein (HDL) metabolism and reverse cholesterol transport (RCT) from macrophages to feces. METHODS AND RESULTS: Experiments were carried out in mice fed a low-fat, low-cholesterol diet, an HFHC diet, or an HFHC diet without added cholesterol (high-saturated fatty acid and low-cholesterol [HFLC]). The HFHC diet caused a significant increase in plasma cholesterol, HDL cholesterol, and liver cholesterol and enhanced macrophage-derived [(3)H]cholesterol flux to feces by 3- to 4-fold. These effects were greatly reduced in mice fed the HFLC diet. This HFHC diet-mediated induction of RCT was sex independent and was not associated with obesity or insulin resistance. The HFHC diet caused 1.4- and 3-fold increases in [(3)H]cholesterol efflux to plasma and HDL-derived [(3)H]tracer fecal excretion, respectively. Unlike a low-fat, low-cholesterol and HFLC diets, the HFHC diet increased liver ABCG5/G8 expression. The effect of the HFHC diet on fecal macrophage-derived [(3)H]cholesterol excretion was totally blunted in ABCG5/G8-deficient mice. CONCLUSION: Despite its deleterious effects on atherosclerosis, the HFHC diet promoted a sustained compensatory macrophage-to-feces RCT. Our data provide direct evidence of the crucial role of dietary cholesterol signaling through liver ABCG5/G8 upregulation in the HFHC diet-mediated induction of macrophage-specific RCT.

ATP-binding cassette G5/G8 deficiency causes hypertriglyceridemia by affecting multiple metabolic pathways.

Mutations in ABCG5 or ABCG8 transporters are responsible for sitosterolemia, an autosomal recessive disease characterized by the accumulation of plant sterols. The aim of this study was to investigate the effects of ABCG5 and ABCG8 deficiency on TG metabolism in mice. Experiments were carried out in wild-type (G5/G8+/+) mice, mice heterozygous for ABCG5 and ABCG8 deficiency (G5/G8+/-) and ABCG5/G8-deficient (G5/G8-/-) mice fed a chow diet. Plasma TG were 2.6 and 4.3-fold higher in fasted G5/G8+/- and G5/G8-/- mice, respectively, than in G5/G8+/+ mice. Postprandial TG were 5-fold higher in G5/G8-/- mice. TG metabolism studies indicate that: first, the fractional catabolic rate was significantly lower in G5/G8+/- (1.3-fold) and G5/G8-/- mice (1.5-fold) compared to G5/G8+/+ and postheparin plasma lipoprotein lipase activities were significantly lower in G5/G8+/- (1.8-fold) and G5/G8-/- mice (5.4-fold) than in G5/G8+/+. Second, liver TG secretion was 1.3-fold higher in G5/G8+/- and G5/G8-/- than in G5/G8+/+ mice and this was associated with an increase in liver LXR, FAS, ACAC and CD36 gene expression. Third, TG intestinal secretion, determined after an oral fat gavage of glycerol tri[9,10(n)-(3)H] oleate, was 5.8-fold higher in G5/G8-/- than in G5/G8+/+ mice. Also, the HOMA index was 2.6-fold higher in G5/G8-/- than in G5/G8+/+ mice, reflecting a degree of insulin resistance. In conclusion, ABCG5/G8 deficiency in mice fed a chow diet markedly raises TG levels by impairing TG catabolism and by increasing liver and intestinal TG secretion.

[Vitamin treatments that lower homocysteine concentration: can they decrease cerebrovascular disease in primary prevention?]. / Tratamientos vitaminicos para disminuir la concentracion de homocisteina: reducen el riesgo de enfermedad cerebrovascular en prevencion primaria?

INTRODUCTION: High plasma homocysteine (Hcy) concentration or hyperhomocysteinemia is associated with an increased vascular risk of disease in case-control studies and, to a lesser extent, in prospective studies. DEVELOPMENT: Several large randomized, double-blind, placebo-controlled trials have been already conducted using specific vitamin therapies with the aim of reducing secondary cardiovascular (HOPE, NORVIT, WAFACS and WENBIT studies) and cerebrovascular (VISP study) disease risk. The results from these major secondary prevention trials and one meta-analysis, that included other smaller studies up to 12 of them, showed that treatment decreased plasma Hcy concentration but failed to reduce cardiovascular risk. It is nevertheless noteworthy that a recent meta-analysis addressing the effects of these vitamin treatments on cerebrovascular risk found a positive effect on primary stroke prevention. These data would be consistent with the fact that increased Hcy is known to be associated more strongly with stroke risk than with cardiovascular risk. Moreover, folic acid supplementation in grain food has recently been shown to be associated with a decreased stroke incidence in USA and Canada. CONCLUSIONS: Obviously, these data on primary stroke prevention will require extensive confirmation. However, there now appear to be more reasons to expect a positive outcome of Hcy intervention studies.

Aplicación de un panel de metilación para el diagnóstico de cáncer de pulmón en broncoaspirados. Resultados preliminares / Application of a methylation panel for the diagnosis of lung cancer in bronchoaspirates. Preliminary results

Introducción. La metilación del ácido desoxirribonucleico (ADN) es una rama de la epigenética que puede ser útil para la identificación, incluso precoz, del cáncer de pulmón. El objetivo del estudio fue evaluar la sensibilidad, especificidad y rendimiento diagnóstico de un panel de metilación formado por los genes APC (adenomatous polyposis coli), DAPK (death associated protein kinase) y RASSF1A (Ras association domain familiy 1A) asociado a la citología habitual en broncoaspirados (BAS) de pacientes con sospecha de cáncer de pulmón. Material y métodos. Se seleccionaron 39 BAS, 24 positivos y 15 negativos, para cáncer de pulmón de diferentes tipos y estadios. Las citosinas de las muestras se transformaron en uracilos con bisulfito sódico, se efectuó una doble reacción en cadena de la polimerasa (PCR) (bisulfite conversion specific-methylation specific PCR) y se identificó el estado de metilación mediante electroforesis. Resultados. APC resultó metilado en 2 de los 24 tumores pulmonares (8%), DAPK en 0 de 24 (0%) y RASSF1A en 9 de 23 (39%). La sensibilidad total del panel fue del 37,5% (9 de 24). No se detectó metilación en ninguna de las muestras libres de cáncer de pulmón (0 de 15), por lo que la especificidad fue del 100%. Además, el panel detectó metilación en una de las 7 muestras de cáncer primario de pulmón, cuya prueba citológica había sido negativa (14%) y en 3 de las 5 muestras con cáncer de pulmón con citologías meramente sospechosas (60%). Su contribución al diagnóstico correcto ante pruebas citológicas sospechosas o discordantes con el diagnóstico final fue del 33% (4 de 12). Conclusiones. La metilación del ADN puede ser un arma útil en el diagnóstico del cáncer de pulmón. Hasta el momento, la sensibilidad en el panel escogido depende exclusivamente de RASSF1A. Nuevos estudios son necesarios para garantizar la reproducibilidad y optimizar su sensibilidad (AU)

Introduction. DNA methylation is a part of epigenetics that can be useful for lung cancer detection even at early stages. The aim of this study was to evaluate sensitivity, specificity and diagnostic capacity of a methylation panel including APC, DAPK and RASSF1A genes, together with routine cytology tests in patients suspected with lung cancer. Material and methods. We selected 39 bronchoaspirates, 24 positive and 15 negative for lung cancer of different histological types and stages. Samples were transformed with sodium bisulphite. A double PCR (BS-MSP) was performed and methylation status was identified by electrophoresis. Results. APC was found to be methylated in 2 out of 24 lung tumours (8%), DAPK in 0 out of 24 (0%) and RASSF1A in 9 out of 23 (39%). Panel sensibility was 37.5% (9/24). No methylation was detected in any of the negative lung cancer samples (0/15), so specificity was 100%. Moreover, the panel detected methylation in 1 of the 7 primary lung cancer tumour samples in which the cytology test was negative (14%) and in 3 of 5 lung cancer tumour samples in which cytology test was only suspicious (60%). Its contribution to the correct diagnosis when cytology tests were suspicious or discordant with final diagnosis was 33% (4/12). Conclusions. DNA methylation can be a useful tool in lung cancer diagnosis. The chosen panel sensibility depends exclusively on RASSF1A to date. Consequently, new studies are required to guarantee reproducibility and optimize sensitivity (AU)

Increased plasma levels of plant sterols and atherosclerosis: a controversial issue.

A number of studies have raised the possibility of circulating plant sterols being a risk factor in the pathogenesis of atherosclerosis. Evidence in support of this hypothesis comes mainly from observations in sitosterolemic patients, who hyperabsorb plant sterols and suffer premature atherosclerosis. Accordingly, the atherogenicity of plant sterols of dietary origin is currently under debate, in view of the widespread use of cholesterol-lowering functional foods enriched with these compounds. Although some reports have suggested the vascular perils of small increases in plasma plant sterol concentrations, other prospective and large population-based studies have indicated otherwise. Further, the potential risk of plant sterol-enriched foods may be counterbalanced by the notable reduction in plasma cholesterol. This review summarizes the current evidence on the possible impact of plant sterols as a risk factor for atherosclerosis.

El laboratorio clínico y las dislipemias / The clinical laboratory and dyslipidemia

Las dislipemias (alteraciones del metabolismo lipoproteínico) son un factor de riesgo de enfermedad cardiovascular de creciente prevalencia. La determinación de las concentraciones de lípidos, lipoproteínas y proteínas relacionadas circulantes permite el diagnóstico, el tratamiento y el seguimiento de las diferentes dislipemias. Sin embargo, existen numerosos factores que influyen en la concentración de estos constituyentes en la circulación y dependen tanto del individuo (hábitos, características físicas, otros factores de riesgo) como de la muestra analizada y la metodología analítica empleada, y hay que tenerlos en cuenta a la hora de valorar e interpretar los resultados analíticos. Asimismo, las diferentes dislipemias pueden tener su origen no sólo en factores ambientales, sino en factores genéticos; por este motivo, la inclusión de pruebas genéticas para el diagnóstico de las dislipemias es cada vez más frecuente Objetivo: Conocer el proceso de adaptación a la diabetes mellitus tipo 1 (DM1) y analizar su correspondencia con las etapas del proceso de duelo descritas por Kübler-Ross. Sujetos y método: Estudio etnográfico mediante entrevistas en profundidad a 20 pacientes, 10 familiares y 12 profesionales (6 médicos y 6 enfermeras). Para el análisis se siguió el esquema de análisis de datos cualitativos de Miles y Huberman. Resultados: El paciente diagnosticado de DM1 y su familia afrontan la pérdida del estilo de vida y los objetos reales o imaginarios de su vida pasada. Enfermos y familiares experimentan reacciones emocionales que, en algún caso, pueden asemejarse a las etapas de duelo descritas por Kübler-Ross en una enfermedad terminal (negación, rebeldía, negociación, depresión y aceptación), pero hay diferencias que dependen de factores personales y psicosociales. Los profesionales tienden a relacionar la mala adherencia con la negación de la enfermedad, pero algunos pacientes se sienten amenazados por las exigencias de tratamiento y control y por sus consecuencias en su calidad de vida, y conscientemente optan por no seguir las recomendaciones. Es más realista hablar de adaptación a la enfermedad que de aceptación, puesto que los procesos de pérdida son constantes y el enfermo debe reconstruir nuevas identidades según su estado. El proceso de duelo afecta también a la familia y puede ser diferente que el del enfermo en tiempo, intensidad y valoración de los problemas. Conclusiones: La adaptación es un proceso complejo en el que intervienen muchas variables. Se observan diferencias en los mecanismos que utiliza cada sujeto en particular. Los profesionales sanitarios y, particularmente la enfermera, deben considerar las múltiples dimensiones psicosociales de la enfermedad crónica (AU)

The prevalence of dyslipidemia (alterations in lipoprotein metabolism) as a risk factor for cardiovascular disease continues to rise. Determination of circulating levels of lipids, lipoproteins and related proteins allows the diagnosis, treatment and follow-up of the various types of dyslipidemia. However, numerous factors influence the concentration of these constituents in the circulation. These factors depend both on the individual (habits, physical characteristics, presence of other risk factors) and on the sample and analytical methods employed and must be taken into account when assessing and interpreting laboratory results. Similarly, the cause of the different forms of dyslipidemia may lie not only in environmental but also in genetic factors. Consequently, the inclusion of genetic tests in the diagnosis of dyslipidemia is increasingly (..) (AU)

The clinical laboratory and dyslipidemia.

The prevalence of dyslipidemia (alterations in lipoprotein metabolism) as a risk factor for cardiovascular disease continues to rise. Determination of circulating levels of lipids, lipoproteins and related proteins allows the diagnosis, treatment and followup of the various types of dyslipidemia. However, numerous factors influence the concentration of these constituents in the circulation. These factors depend both on the individual (habits, physical characteristics, presence of other risk factors) and on the sample and analytical methods employed and must be taken into account when assessing and interpreting laboratory results. Similarly, the cause of the different forms of dyslipidemia may lie not only in environmental but also in genetic factors. Consequently, the inclusion of genetic tests in the diagnosis of dyslipidemia is increasingly frequent.