M. Anzola

Gene promoter hypermethylation in oral rinses of leukoplakia patients—a diagnostic and/or prognostic tool?

Leukoplakia is the most frequent oral precancerous lesion and shows a variable rate of malignant transformation. We hypothesised that the detection of molecular alterations, like the promoter hypermethylation of DNA, in oral cytological samples from patients at risk of developing primary or recurrent tumours could be a valuable diagnostic and prognostic tool in the management of these lesions. Two groups of patients with differing risks of developing oral squamous cell carcinoma (OSCC) were analysed. DNA was extracted from the oral rinse of each patient. The methylation status of the p16, p14 and MGMT gene promoters was determined using a methylation-specific polymerase chain reaction (MSP). Methylation of p16 and MGMT was observed in 44 and 56% of the oral samples, respectively. Only 12% of the cases showed p14 methylation. DNA hypermethylation was more frequent in patients with previous OSCC. DNA promoter hypermethylation is frequent during early oral carcinogenesis and even more so in the later stages. MSP using oral rinses is a non-invasive and highly sensitive technique which could be used to monitor patients with precancerous and cancerous oral lesions.

Frequent loss of p53 codon 72 Pro variant in hepatitis C virus-positive carriers with hepatocellular carcinoma

Codon 72 exon 4 polymorphism of the p53 gene has been implicated in cancer risk and it has been suggested that it may have an impact on the clinical outcome of the disease. Our objective was to evaluate the association between p53 polymorphism at codon 72 and hepatocellular carcinoma. The p53 codon 72 genotype was examined in 97 biopsy samples from 67 Basque patients histologically diagnosed with hepatocellular carcinoma. Blood samples collected from 111 Basque residents were examined as a control group. The polymorphism was examined by both single strand conformation polymorphism analysis and allele specific polymerase chain reaction. Fishers exact test was used to evaluate the data. The results showed that there were no statistically significant differences in the frequency of codon 72 polymorphism genotype between patients with liver cancer and healthy controls. We found a frequent loss of proline allele in hepatitis C virus (HCV)-positive carriers. In conclusion, the lack of a significant relationship between this polymorphism and risk of hepatocellular carcinoma suggests that it does not predispose towards hepatocarcinogenesis in this population. We suggest that the frequent loss of the proline allele in HCV-associated carcinogenesis of the liver plays some role in hepatocarcinogenesis.

Hepatocellular carcinoma: molecular interactions between hepatitis C virus and p53 in hepatocarcinogenesis

Hepatocellular carcinoma (HCC) is the most important primary hepatic cancer and is a common cancer type worldwide. Many aetiological factors have been related to HCC development, such as liver cirrhosis, hepatitis viruses and alcohol consumption. Inactivation of the p53 tumour suppressor gene is one of the most common abnormalities in many tumours, including HCC. p53 is of crucial importance for the regulation of the cell cycle and the maintenance of genomic integrity. In HCC, hepatitis B and C virus (HBV and HCV) effect carcinogenic pathways, independently leading to anomalies in p53 function. Several authors have reported that some HCV proteins, such as the core, NS5A and NS3 proteins, interact with p53 and prevent its correct function. The mechanisms of action of these HCV proteins in relation to p53 are not completely clear, but they might cause its cytoplasmic retention or accumulation in the perinuclear region where the protein is not functional. The identification of the interactions between p53 and HCV proteins is of great importance for therapeutic strategies aimed at reducing the chronicity and/or carcinogenicity of the virus.

p14arf gene alterations in human hepatocellular carcinoma

Introduction The molecular status of the p14ARF gene has not been fully elucidated in hepatocellular carcinoma (HCC). This study was performed to determine genetic and epigenetic alterations in the p14ARF tumor suppressor gene and their effect on HCC progression. Methods The status of p14 was evaluated in 117 HCC tumoral nodules and 110 corresponding non-tumor tissues by loss of heterozygosity at the 9p21-22 region, homozygous deletions, single strand conformation polymorphism-polymerase chain reaction mutational analysis and methylation-specific polymerase chain reaction. Results The most frequent inactivation mechanism was hypermethylation of the promoter region, which was found in 41.9% of tumor samples and in 19.1% of non-tumor samples. Loss of heterozygosity at the 9p21 region was detected in 27.3% and 10% of tumor and non-tumor tissues, respectively. Homozygous deletions and mutations were less common events in hepatocarcinogenesis. We found 5.9% of the tumor cases with exon 2 homozygous deletions and 3.4% of the cases with mutations. We described a silent mutation in codon 42 of exon 1β for the first time. No association was found between inactivation of p14ARF and clinicopathological characteristics or prognosis. Conclusion We can conclude that p14ARF is frequently and early altered in HCC, being the main cause of inactivation promoter hypermethylation. Our results suggest that the p14ARF gene plays an important role in the pathogenesis of hepatocellular carcinoma.

High levels of p53 protein expression do not correlate with p53 mutations in hepatocellular carcinoma

Summary.  To determine the relationship between p53 altered expression and p53 mutations in hepatocellular carcinoma (HCC), we analysed p53 protein immunohistochemically and assessed the presence of mutations in exons 4–8 of the p53 gene using SSCP assay in 117 HCCs corresponding to 78 patients. We also determined the relationship of p53 expression with cellular proliferation by immunostaining with monoclonal antibodies to Ki‐67. We found significant levels of p53 protein expression in 23.1% of the 117 cases studied, but identified mutations in only 12 cases (10.3%). Only four of the p53‐positive cases had mutations in the regions analysed. Six of the cases that displayed mutations at p53 gene were negative for immunohistochemical analysis (IHC) and two cases showed positive immunoreactivity in the cytoplasm of the cell.

p16INK4A gene alterations are not a prognostic indicator for survival in patients with hepatocellular carcinoma undergoing curative hepatectomy

Background and Aim:  Hepatocellular carcinoma (HCC) is a common malignancy worldwide that is highly associated with chronic hepatitis B or C infection and cirrhosis. The tumor suppressor gene p16INK4A is an important component of the cell cycle and inactivation of the gene has been found in a variety of human cancers. The present study was performed to determine genetic and epigenetic alterations in the p16INK4A tumor suppressor gene and the effect of these on HCC progression.

Patterns of methylation profiles as diagnostic markers for multiple hepatocellular carcinomas.

Background: Hepatocellular carcinoma often displays multiple tumor nodules, thus posing a problem for differential diagnosis between cancers of both multifocal and metastatic origin. Conventionally, pathological criteria have been used for this purpose, but these are largely subjective. In order to facilitate a more objective differential diagnosis of multiple HCCs, we used the patterns of methylation of p16 INK4a , p14 ARF , and GSTP1 genes as markers for each tumor nodule. Methods: Sixty‐seven nodules from 30 cases of multiple or recurrent HCCs were examined using methylation‐specific PCR (MSP) analysis for the detection of methylation profiles. Results: Hypermethylation was detected in 56.7%, 43.3% and 17.9% of the cases for p16 INK4a , p14 ARF , and GSTP1 genes, respectively. At the genetic level the inter‐nodule methylation profiles were heterogeneous in 23 of the cases and homogeneous in another 7, enabling a multifocal origin to be diagnosed in the former and metastatic origin in the latter. Conclusions: Methylation profiling seems to be useful in differentiating the clonal origins of multiple cancers, as the information yielded by this method is essentially objective.

No association between GSTP1 gene aberrant promoter methylation and prognosis in surgically resected hepatocellular carcinoma patients from the Basque Country (Northern Spain)

Aims: Hepatocellular carcinoma (HCC) is one of the most common cancers worldwide, being linked etiologically to several factors. Glutathione‐S‐transferases (GSTs) are a family of enzymes that play an important role in detoxification. Hypermethylation of regulatory sequences at glutathione‐S‐transferase pi class gene (GSTP1) has been found in different human tumor types. In this study, we have studied the methylation status of the GSTP1 promoter region in patients from the Basque Country (Northern Spain) by methylation‐specific PCR (MSP).

p53, un gen supresor tumoral

Resumen Los genes supresores tumorales estan implicados en diversos procesos de division celular como la regulacion de la expresion genica, control del ciclo celular, programacion de la muerte celular y estabilidad del genoma. La perdida de actividad de estos genes provoca la incapacidad de respuesta a los mecanismos de control que regulan la division celular, de modo que se produce una proliferacion mas o menos incontrolada de la celula lo cual conduce en ocasiones al desarrollo de neoplasias y a la evolucion de las mismas hacia procesos tumorales mas agresivos. El gen p53 pertenece al grupo de genes implicados en el control del ciclo celular. Este gen tiene multiples funciones ya que aparece implicado no solo en el control del ciclo celular sino tambien en la integridad del ADN y la supervivencia de las celulas expuestas a agentes que danan el ADN. La alteracion del gen p53 confiere un riesgo muy elevado de desarrollar cancer y la mutacion del mismo es uno de los cambios genomicos mas frecuentes en el cancer humano.