Investigating the role of human papillomavirus in lung cancer.

Lung cancer is the leading cancer worldwide among men and women with morbidity reaching 1.6 million. Human Papillomavirus is the causal factor of cervical cancer while its association with others is still under investigation. The purpose of our study is to examine the presence of HPV DNA as well as high-risk E6/E7 mRNA in patients with lung cancer. Lung tissues were collected during bronchoscopy and tested for HPV DNA and E6/E7 mRNA. 67 lung tissue samples were analysed. The age range was 49-85 years old (y.o) with a mean age of 67.6 y.o. 9 patients were female and 58 were male. The study included 12 Small Cell Lung Cancers (SCLC) and 55 Non Small Cell Lung Cancer (NSCLC). HPV DNA was detected in 3.0% (2/67) of lung cancer cases, while no E6/E7 mRNA of five high-risk HPV types was found in tissue samples examined. The two positive patients had no prior history of an HPV related disease. Using the mRNA test as a gold standard for the association of HPV with malignant transformation, the present results showed no association of HPV status with lung cancer. Further investigation of more lung cancer tissues is required to reach safe conclusions.

Identification of rearranged sequences of HPV16 DNA in precancerous and cervical cancer cases.

Integration of HPV16 DNA into the host chromosome is considered to be a crucial step towards genomic instability and cervical cancer development. Aim of the present study was to investigate the presence of HPV16 rearranged intra-viral sequences in HPV16-positive normal, precancerous and cervical cancer samples using the method of Restriction Site-PCR (RS-PCR). Sequence analysis of HPV16 integrants revealed for the first time in clinical samples two distinct rearranged intra-viral sequences, concerning the conjunction of E2 and L1 genes and the conjunction of E1 and L1 genes with inverted orientation. Furthermore mapping analysis of the E1 and E2 genes in cervical samples with rearranged intra-viral sequences of HPV16 genome was conducted in order to determine the integrity of viral genes. The identification of intra-viral rearrangements provides valuable information regarding the HPV16 DNA integration, and may be a significant biomarker for the presence of chromosomal instability and DNA damages in clinical samples.

Prevalence of HPV16 E1-137463nt variants in Greek women.

Recent studies have focused on sequence variation of the HPV16 E1 gene. The present study investigates the prevalence of E1-1374^63nt duplication in the Greek population, and the sequence variation at the 5' end of the E1 and E6 genes from samples that harbored this genetic alteration. Fifty HPV16 positive cervical samples, derived from Greek patients were investigated. The 5' end of the E1 gene was amplified through PCR and the variant amplicons were cloned, sequenced, and bioinformatically analyzed for selective pressure. The E1-1374^63nt duplication was identified in 24% of the examined samples, with the same prevalence in both high and low-grade cervical malignancies. The E1-1374^63nt duplication was linked to the European variant lineage (x² = 5.076, P < 0.024) and it was significantly associated with the nucleotide variation A1053C (x² = 23.102, P < 0.0001). Molecular evolution analyses anticipate that the E1-1374^63nt duplication induces functional constraints on the 5' end of E1 gene, and it is proposed that this duplication might not affect negatively the function or structure of the E1 protein. The E1-1374^63nt duplication is prevalent in the Greek population, whereas the A1053C variation might constitute a significant marker for the characterization of the E1-1374^63nt variant in the Greek population, thus providing significant information about viral pathogenicity.

Identification of novel E6-E7 sequence variants of human papillomavirus 16.

The rate of evolution of the human papillomavirus 16 (HPV16) genome is low. However, the ability of the E6 oncoprotein to interact with distinct p53 variants causes selective pressure on the E6 gene. In addition, intratypic recombination events in the HPV16 E6 and E7 genes have been characterized as extraordinary phenomena during the evolutionary history of virus. In the present study, we identified two new sequence variants through nucleotide analysis of the E6-E7 region of the HPV16 genome. Maximum-likelihood and empirical Bayesian methods were used in order to identify positive selection at particular residues of the E6 and E7 genes. Using the single recombination breakpoint (SBP) method, we found evidence of recombination events in the E6 ORF. Nucleotide sequence analysis showed that the new sequence variants are phylogenetically distant from the other members of the population. Our results indicate that new evolutionary intermediates of HPV16 might be formed either though positive selective pressure or through recombination events by multiple infections with distinct HPV16 variants.

Sequence variation analysis of the E2 gene of human papilloma virus type 16 in cervical lesions from women in Greece.

The E2 gene of human papilloma virus is expressed at the early stage of the viral life cycle, encoding the E2 transcription factor, and regulates the expression of E6 and E7 oncogenes. Disruption of E2 gene due to viral integration inhibits the transcriptional suppression of the HPV oncogenes, inducing cell proliferation. In the present study, a total of 22 HPV16-positive cytological specimens derived from high- and low-grade cervical intraepithelial lesions were investigated in order to identify sequence variations in the HPV16 E2 ORF. The E2 gene was amplified by PCR using external and internal overlapping sets of primers. Amplicons were cloned and sequenced. Disruption sites were detected in cervical samples diagnosed as high-grade cervical intraepithelial lesions. Moreover, sequence variations were identified in the E2 ORF and specific variations were associated with non-European variants such as African type I, African type II and Asian American. A total of three new sequence variations were identified at positions 2791, 2823 (transactivation domain) and 3361 (hinge region). Distinct phylogenetic branches were formed according to E2 analysis that characterized the different HPV16 variants. It was ascertained that non-European variants are circulating in the Greek population.

Alteration of integrin expression in oral squamous cell carcinomas.

OBJECTIVE: This study examines the intensity of expression of beta 1, alpha 2, alpha 3, alpha 5, alpha 6 integrin subunits in oral squamous cell carcinoma (SCC) as opposed to normal oral epithelium, and the intensity of expression and distribution pattern of the above subunits in relation to tumour differentiation grade. MATERIALS AND METHODS: Cryostat sections of 25 cases of oral SCC and 15 cases of normal oral epithelium were studied by immunohistochemistry (APAAP method). RESULTS: The intensity of expression of beta 1, alpha 2 (Pearson chi 2 P < 0.001) and alpha 6 (Test for Trend P < 0.05) integrin subunits was reduced significantly in SCC compared to normal oral epithelium. All integrin subunits were mainly expressed in the peripheral cell layer of tumour islands. No correlation was found between the intensity of integrin expression and the degree of differentiation in SCC. The same applied to the distribution pattern of the integrin subunits. By means of cross examination of all integrins, the loss of intensity of alpha 2 beta 1 integrin expression was found to have the strongest correlation with oral SCC (Ordered Logistic Regression). CONCLUSIONS: Reduced intensity of expression of all subunits was found in oral SCC compared to normal epithelium. Further investigation is needed to determine whether alpha 2 beta 1 integrin expression can be used as a prognostic evaluator for the behaviour of the disease.

Isolation of a cDNA clone from colon carcinoma.

cDNA clones of differentially expressed mRNAs in a colon carcinoma have been isolated by subtractive cDNA cloning. The substracted material was at least 90x enriched for differentially expressed sequences and can be used for the construction of substractive cDNA libraries and polymerase chain reaction (PCR) amplification to generate differential probes. In this way rare mRNA (less than 0.1% abundance) which are differentially expressed, can be isolated utilising this procedure. A cDNA clone which we call IH12 has been isolated. Its mRNA is expressed exclusively in actively proliferating cells.

Human papillomaviruses in cervical cancer I. HPV-16 and 18 predominate in the Greek population.

Human papillomaviruses (HPV) and their role in carcinogenesis have been the subject of extensive investigation Specific types of HPV have been associated with cervical carcinoma HPV 16 and 18 are mainly associated with malignant progression and considered "high risk" viruses Using Southern blot analysis and in situ hybridization we investigated the presence of papilloma viruses in cervical carcinoma patients as well as appropriate controls. The results presented here support the aetiological role of HPV 16 and 18 in cervical carcinoma and demonstrate the prevalence of these viruses in Greek women. The role of viruses in carcinogenesis in well established in almost all species from fishes, to birds, to mammals. Although not well circumstantiated, viruses probably play as-great a role in human cancer as in other species. The role of human papillomaviruses (HPV) not only in benign proliferations, but also in a number of malignancies has long been postulated (1,2). Presently over 20 HPV types have been identified and there is evidence now associating specific types with certain human anogenital cancers, notably cervical cancer (3,4). Advance neoplasias such as squamous cell carcinomas are associated with types, 16,18 and 31, with type 16 prevailing in these lesions (5,6). In this paper we shall present evidence which extends and confirms that previously reported on the prevalence of HPV 16 and 18 in Greek women.

Human papillomaviruses in cervical cancer: II. Application of PCR and in situ hybridization and HPV significance.

Human papillomaviruses especially type 16 and 18 are associated with cervical cancer. These viruses encode transforming proteins which are capable of binding and form complexes with p53 protein. Tumour-tissue samples from women with cervical cancer as well as normal controls were analysed for the presence of HPV and the expression of p53 by in situ hybridization and PCR analysis. The results showed that HPV 16 and 18 predominated in all pathological conditions and that p53 expression was related to the presence of certain types of HPV. The significance of HPV in cervical carcinoma is well demonstrated.

Nasopharyngeal carcinoma: Epstein-Barr Virus significance.

Thirty-one cases of NPC were investigated by in situ hybridization, PCR analysis and serology to assess the presence of EBV and to interrelate these findings. Statistical analysis showed a significant correlation between the probability of detecting EBV and NPC. There was no significant association between in situ hybridization, PCR and serology. Tumor size correlated with EBNA, but no correlation between smoking and NPC was found.

Establishment and characterization of a hepatoblastoma cell-line from a patient with secondary acute myeloid-leukemia.

A girl who had been treated, apparently successfully, with surgery and chemotherapy for a hepatoblastoma, fell ill two years later with what was diagnosed as an AMF M(4). A cell line was established from her peripheral blood. This cell line had epithelial morphology and grew both in suspension culture and as a monolayer. The cells were positive for epithelial surface markers, including the liver-specific alpha-fetoprotein, but not for leukocyte markers. The cell-line's karyotype was markedly abnormal. It did not have any specific aneuploidies or any other aberrations characteristic of leukemias; instead it had gains of 2q and chromosome 20, the most common cytogenetic changes in hepatoblastoma. It is most likely that the patient had a relapse of hepatoblastoma with massive seeding of the blood leading to a leukemia-like picture without, of course, excluding other possibilities.

Relative paucity of p53 gene-mutations in male breast carcinomas.

Mutations of the p53 suppressor -ene are the most common genetic lesion noted in human cancers and appear to be relatively common (30%) as somatic cell mutations in female breast cancer. p53 mutations have also been frequently reported in familial breast cancers as in Li-Fraumeni syndrome (LFS). Males with breast cancer are far rarer than females. We investigated the mutational spectra of the p53 gene in male breast cancers. Of 10 samples analyzed for p53 mutations in exons 5, 6. 7 and 8, only two showed point mutations corresponding to amino acid residues 248 and 290. One of the point mutations turned out to be a silent change, thus representing only DNA polymorphism. Although the number of male breast cancer samples thus far examined is small, the p53 mutations in male breast cancer (10%), unlike females (30%), does not appear to be as frequent.

Subtraction hybridization cDNA libraries from colon carcinoma and hepatic cancer.

cDNA clones of differentially expressed mRNAs in a colon carcinoma and a hepatocellular carcinoma have been isolated by subtractive cDNA cloning. The subtracted material is at least 90 X enriched for differentially expressed sequences and can be used for construction of subtractive cDNA libraries and polymerase chain reaction (PCR) amplification to generate differential probes. Commercially available lambda ZAP II is used for construction of primary libraries since single-stranded phage bearing the cloned cDNA can be excised in vivo and because lambda libraries are convenient for subsequent screening and manipulations. Rare mRNAs (less than 0.01% abundance), which are differentially expressed, can be isolated utilizing this procedure.