Nerea Cuevas

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.

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).

Cytochrome b and HVI sequences of mitochondrial DNA to identify domestic animal hair in forensic casework

Biological traces that appear at the scene of a crime or on the body of the victim may be of human, animal and/or vegetable origin. Among those of animal origin, household pets are a common source, with pet hair being one of the most frequent traces found. Consequently, it is necessary to have laboratory methods capable of identifying traces from pets and domesticated animals in general. Savolainen et al. [1] have developed a basic method of sequencing the HVI region of mitochondrial DNA from Canis familiaris using single hairs as template, with a discrimination capacity of 1 in 10 individuals. The species from which a biological trace has come can be identified by analyzing a short fragment of the cytochrome b (cytb) gene sequence of the mitochondrial genome. This gene contains species-specific information and has been used in phylogenetic as well as in forensic investigations in a number of studies. Parson et al. [2] have confirmed the usefulness of cytb analysis in identifying the biological origin of casework specimens.