Maria Luisa Maestro

Relation of IL28B gene polymorphism with biochemical and histological features in hepatitis C virus-induced liver disease.

Background/Aims Polymorphism at the IL28B gene may modify the course of hepatitis C virus (HCV) chronic infection. Our aim was to study the influence of IL28B rs12979860 gene polymorphism on the biochemistry and pathology of HCV-induced disease in the clinical course from mild chronic hepatitis C to hepatocellular carcinoma. Methods We have determined the rs12979860 single nucleotide polymorphism (SNP) upstream IL28B gene in two groups of patients with HCV-induced chronic liver disease: 1) 268 patients (159 men) with biopsy-proven chronic hepatitis C, to analyse its relation with biochemical, virological and histological features; and 2) 134 patients (97 men) with HCV-related hepatocellular carcinoma. The distribution of the analysed SNP in hepatocellular carcinoma patients was compared with that found in untreated chronic hepatitis C patients. All patients were white and most were Spaniards. Results In multivariate analysis ALT values were higher (P = 0.001) and GGT values were lower (P<0.001) in chronic hepatitis C patients homozygotes for the major rs12979860C allele as compared with carriers of the mutated rs12979860T allele. Steatosis was more frequent (Odds ratio = 1.764, 95% C.I. 1.053–2.955) and severe (P = 0.026) in carriers of the rs12979860T allele. No relation was found between the analysed SNP and METAVIR scores for necroinflammation and fibrosis, and there were no differences in the distribution of the analysed SNP between hepatocellular carcinoma and untreated chronic hepatitis C patients. Conclusion The IL28B rs12979860 polymorphism correlates with the biochemical activity and the presence and severity of liver steatosis in chronic hepatitis C.

Prognostic significance of p16INK4a alterations and 9p21 loss of heterozigosity in locally advanced laryngeal squamous cell carcinoma

The p16INK4a gene, localized within chromosome 9p21, has been identified as a cyclin‐dependent kinase inhibitor and may negatively regulate the cell cycle acting as a tumor suppressor. Genetic alterations involving the 9p21 region are common in human cancers. A consecutive series of 64 untreated patients (median of follow up 53 months) undergoing surgical resection for locally advanced laryngeal squamous‐cell carcinomas (LSCCs) has been studied prospectively. Our purpose was to investigate p16 alterations (9p21 allelic loss, hypermethylation and point mutations) and their possible association with clinico‐pathological data and flow cytometric variables (DNA‐ploidy and S‐phase fraction (SPF)), and to determine the possible prognostic role of this gene in these tumors. PCR‐based techniques were used for investigating 9p21 loss of heterozygosity (LOH) and methylation promoter status of the p16 gene. p16 mutations were detected by PCR‐SSCP (single strand conformation polymorphism) and sequencing. 9p21 LOH was detected in 16/62 (26%) informative tumors, point mutations in 5% (3/64) and hypermethylation in 9% (6/64) of the cases. p16 alterations were significantly associated with high SPF and DNA‐aneuploidy. By univariate analysis, poor histologic differentiation, stage IV, DNA‐aneuploidy and p16 point mutations proved to be significantly related to quicker relapse, whereas these same factors, and in addition high SPF, 9p21 LOH and any p16 alterations were significantly related to shorter overall survival. By Cox proportional hazards analysis only histologic grade (G3) and p16 point mutations were independently related to both disease relapse and death. Our study has identified p16 point mutations as important biomolecular indicators in LSCCs.

Influence of vitamin D-related gene polymorphisms (CYP27B and VDR) on the response to interferon/ribavirin therapy in chronic hepatitis C.

Background and Aims Vitamin D exerts immunomodulatory effects on the host response against infection with hepatitis C virus (HCV). This study was performed to assess the putative influence of polymorphisms in vitamin D-related genes on the response to antiviral therapy in patients with chronic hepatitis C (CHC). Methods Single nucleotide polymorphisms (SNPs) in CYP27B-1260 gene promoter (rs10877012AC) and in vitamin D receptor (VDR) gene rs2228570TC, rs1544410CT, rs7975232AC and rs731236AT were analyzed in a cohort of 238 Caucasian CHC patients treated with pegylated interferon (Peg-IFN) plus ribavirin (RBV). Multivariate analyses were performed to exclude confounding effects of well-known baseline predictors of response to therapy (HCV genotype and load, IL28B genotype, age, and GGT and serum cholesterol). Results Three SNPs at the VDR gene (rs1544410, rs7975232 and rs731236) were in strong linkage disequilibrium, with the CCA haplotype predicting therapeutic failure [Odds ratio 2.743; (95% C.I. 1.313–5.731), p = 0.007]. The carrier state of the VDR rs2228570 T allele was inversely related to the probability of therapeutic failure [Odds ratio 0.438; 95 C.I. (0.204–0.882), p = 0.021]. No relation existed between CYP27B-1260 rs10877012 polymorphism and response to therapy. The area under the operating curve (AUROC) based on the model including all variables significantly related to the response to therapy was 0.846 (95% confidence interval = 0.793–0.899). Conclusion VDR gene polymorphisms are independently related to the response to Peg-IFN+RBV therapy in chronic hepatitis C and could be used as complementary biomarkers of response when included in a prediction algorithm in association with demographic, virologic, biochemical and genetic traits.

Serum level of fibroblast growth factor 23 in maintenance renal transplant patients

BACKGROUND The discovery of fibroblast growth factor 23 (FGF23) provides a new conceptual framework that improves our understanding of the pathogenesis of post-transplant bone disease. Excess FGF23 is produced in the early post-transplant period; levels return to normal in the months following transplant. However, few manuscripts discuss FGF23 levels in stable long-term renal transplant recipients. METHODS We performed a cross-sectional observational study of 279 maintenance kidney recipients with chronic kidney disease (CKD) Stages 1-4 and stable allograft function who had received their transplant at least 12 months previously. We calculated the estimated GFR (eGFR) using the MDRD4 equation. RESULTS FGF23, parathyroid hormone (PTH) and phosphorus values were higher in more advanced stages, while the serum calcitriol levels and the phosphate reabsorption rate were lower. A significant inverse correlation was found between eGFR and FGF23 (r = -0.487; P < 0.001), PTH (r = -0.444; P < 0.001), serum phosphate levels (r = -0.315; P < 0.001) and fractional excretion of magnesium (r = -0.503; P < 0.001). Multivariable analysis showed that increased time on corticosteroids (P < 0.001), PTH (P < 0.001), serum phosphate (P = 0.003), decreased serum calcitriol (P = 0.049) and estimated glomerular filtration (P = 0.003) rate were associated with high FGF23 levels. In contrast with pre-transplant patients and first year post-transplant patients, higher FGF23 values were not correlated with increased phosphate excretion. An elevated phosphate reabsorption rate was associated with decreased PTH (P < 0.001) and calciuria (P = 0.028) and increased serum calcitriol (P = 0.009), plasma bicarbonate (P = 0.024) and estimated glomerular filtration (P = 0.003). CONCLUSIONS Serum FGF23 concentrations remain increased in long-term kidney graft recipients, even in the early stages of CKD. It remains to be seen whether measures aimed at reducing serum levels of PTH and phosphate and/or corticosteroid doses might help to lower serum FGF23 and whether this will improve kidney recipient outcomes.

seqCNA: an R package for DNA copy number analysis in cancer using high-throughput sequencing

BackgroundDeviations in the amount of genomic content that arise during tumorigenesis, called copy number alterations, are structural rearrangements that can critically affect gene expression patterns. Additionally, copy number alteration profiles allow insight into cancer discrimination, progression and complexity. On data obtained from high-throughput sequencing, improving quality through GC bias correction and keeping false positives to a minimum help build reliable copy number alteration profiles.ResultsWe introduce seqCNA, a parallelized R package for an integral copy number analysis of high-throughput sequencing cancer data. The package includes novel methodology on (i) filtering, reducing false positives, and (ii) GC content correction, improving copy number profile quality, especially under great read coverage and high correlation between GC content and copy number. Adequate analysis steps are automatically chosen based on availability of paired-end mapping, matched normal samples and genome annotation.ConclusionsseqCNA, available through Bioconductor, provides accurate copy number predictions in tumoural data, thanks to the extensive filtering and better GC bias correction, while providing an integrated and parallelized workflow.

Detección y cuantificación de células tumorales en sangre periférica en pacientes con cáncer de colon

BACKGROUND AND OBJECTIVE: The aim of this study is the detection and quantification of circulating tumor cells (CTC) in patients diagnosed with colon cancer and to establish whether they are related to the main clinicopathologic variables for this type of carcinoma. PATIENTS AND METHOD: Twenty-five colon cancer patients and 30 healthy volunteers were analysed. The quantification was performed using the CellSpotter Analyzer (Veridex LLC), that allows immunomagnetic isolation and immunospecific labelling of the cells for their enumeration. RESULTS: 72% of the colon cancer patients showed CTC and the mean number of cells found was 5 CTC/7.5 ml of peripheral blood. 52% of the samples contained 2 or more cells. Considering 2 cells as the cut-off point, a significant relationship with lactate dehydrogenase was found. CONCLUSIONS: This new technology which allows isolation and quantification of CTC in peripheral blood has proven to be valid for the detection of epithelial cells in colon cancer patients in every tumor stage. The results shown in this work confirm that cytokeratin 8, 18 and 19 are detected in CTC in this tumor type and will allow us to develop a protocol for the study of the relationship of quantification of theses cells and the clinical parameters involved in colon cancer.

La inestabilidad de microsatélites predice un mejor pronóstico en los pacientes intervenidos de carcinoma colorrectal

Fundamento y objetivo: En el carcinoma colorrectal se describen 2 vias geneticas diferentes implicadas en la genesis del tumor: la inestabilidad cromosomica, debida a la alteracion de genes supresores o protooncogenes, y la inestabilidad de microsatelites, originada por alteraciones en los genes reparadores del ADN. Pacientes y metodo: En este estudio se determina la frecuencia y el significado clinico de la via de la inestabilidad de microsatelites en una cohorte prospectiva consecutiva de 106 pacientes intervenidos por carcinoma colorrectal por un mismo cirujano. Para la determinacion de la inestabilidad de microsatelites se han seguido los criterios propuestos por el National Cancer Institute en 1998. Resultados: El 9,4% de los pacientes muestra una alta inestabilidad y el 11,3% una inestabilidad baja. Ambos grupos presentan diferentes caracteristicas clinicopatologicas (edad, sexo, localizacion del tumor y tipo histologico). En el analisis multivariante de la supervivencia global y de la supervivencia libre de enfermedad, la alta inestabilidad presenta un valor pronostico independiente del resto de las variables clinicopatologicas analizadas (p < 0,0001). Conclusiones: La alteracion genetica que supone la alta inestabilidad de microsatelites confiere a los pacientes con cancer colorrectal un mejor pronostico.

Effect of β-catenin alterations in the prognosis of patients with sporadic colorectal cancer

CONTEXT Wnt pathway activation represents a critical step in the etiology of most of colorectal cancer (CRC) and it is commonly due to mutations in the APC gene, which originates the loss of β-catenin regulatory function. It has been suggested that APC inactivation or β-catenin alteration have similar effects in tumor progression in CRC tumorigenesis. AIMS The aim of this study was to analyze the frequency of β-catenin gene mutation in patients with sporadic CRC and to determine its effect in prognosis. MATERIALS AND METHODS This was a prospective cohort study, which included 345 patients with sporadic CRC. β-Catenin gene mutations in exon 3 were detected by single strand conformation polymorphism (SSCP). Exon 3 deletion was studied by identifying differences in fragment length of specific amplification products. All the altered samples were confirmed by direct sequencing. RESULTS In our population, point mutations were detected in 1.8% of the samples and 4.9% of the samples showed deletion. We observed association between exon 3 mutations and increased levels of Carcinoenbryonic Antigen (CEA). In these patients, clinically relevant improvement in overall survival was also observed. CONCLUSION Frequency of point mutations in exon 3 β-catenin gene is low in our population. It would be interesting to increase the population size to test the clinically relevant influence in the prognosis found, and to test the relation of these events with Microsatellite Instabillity (MSI) pathway. If these findings were confirmed, β-catenin determination would help in the selection of patients with different prognosis.