Molecular backgrounds of ERAP1 downregulation in cervical carcinoma

The antigen processing machinery (APM) plays an important role in immune recognition of virally infected and transformed cells. Defective expression of the APM component ERAP1 is associated with progression and poor clinical outcome in cervical carcinoma. However, the underlying mechanisms of ERAP1 protein downregulation remain to be established. We investigated ERAP1 mRNA expression levels in 14 patients with established ERAP1 protein downregulation. To further examine the possible pretranscriptional mechanisms of ERAP1 downregulation, ERAP1 DNA mutation status was analyzed alongside existing data on various single nucleotide polymorphisms. Moreover, loss of heterozygosity at various loci in the ERAP1 gene was investigated. In cases with ERAP1 protein downregulation, ERAP1 mRNA quantities were found to be significantly lower than in a cohort with normal ERAP1 protein expression (P = 0.001). Loss of heterozygosity was demonstrated to occur in up to 50% of tumors with ERAP1 downregulation. Our data indicate that ERAP1 downregulation is associated with loss of heterozygosity. These data provide the first insight into in vivo mechanisms of ERAP1 downregulation in cervical carcinoma

Physical status of multiple human papillomavirus genotypes in flow-sorted cervical cancer cells

Multiple human papilloma virus (HPV) infections have been detected in cervical cancer. To investigate the significance of multiple HPV infections, we studied their prevalence in cancer samples from a low-risk (Dutch) and a high-risk (Surinamese) population and the correlation of HPV infection with tumor cell aneuploidy. SPF 10 LiPA was used for HPV detection in formalin-fixed cervical carcinoma samples from 96 Dutch and 95 Surinamese patients. Samples with HPV type 16 or 18 infections were sorted by flow cytometry, and fluorescence in situ hybridization was performed on the diploid and aneuploid subpopulations to detect HPV 16 and 18 genotypes simultaneously. Multiple HPV infections were present in I I of 80 (13.8%) Dutch and 17 of 77 (22. 1%) Surinamese carcinomas. Three cases had an HPV 16 and HPV 18 coinfection: in two cases, integrated HPV copies of HPV 16 or 18 were detected in the aneuploid fraction, and in one case both HPV 16 and 18 were present solely as episomes. Based on our findings, multiple HPV infections are present in cervical cancer samples from both high- and low-risk populations. Furthermore, multiple HPV types can be present in an episomal state in both diploid and aneuploid tumor cells, but integrated HPV genomes are detectable only in the aneuploid tumor cell subpopulations. (c) 2007 Elsevier Inc. All rights reserved

Human papillomavirus type 16 E6, E7, and L1 variants in cervical cancer in Indonesia, Suriname, and the Netherlands

Objective. Human papillomavirus type 16 (HPV 16) has several intratypic variants, and some are associated with enhanced oncogenic potential. For risk determination as well as for future vaccine development, knowledge about variants is important. Regarding the geographical distribution of HPV variants and the lack of data from Indonesia and Suriname, we studied the prevalence of HPV 16 variants in cervical cancer in these high incidence countries. Data were compared with The Netherlands, a low-risk country. Methods. DNA samples from 74 formalin-fixed paraffin-embedded HPV 16-positive cervical carcinomas from Indonesia (Java, N = 22), Suriname (N = 25), and The Netherlands (N = 27) were amplified using primers specific for the E6, E7, and part of the L1 regions. Products were sequenced and analyzed. Results. A specific Javanese variant, with mutations 666A in E7 and 6826T in L1, was found in 73% of the Indonesian samples, 56% having an additional mutation in the E6 open reading frame (ORF; 276G), giving the predicted amino acid change N58S. This Javanese variant was also found in three Surinamese samples, which reflects what could be expected from migration of Javanese people to Surinam. Other non-European variants were identified in Indonesian, Surinamese, and Dutch samples in 14%, 28%, and 19%, respectively. Conclusion. The majority of the HPV 16-positive cervical cancers in Indonesia are caused by a specific intratypic variant that was rarely found before in other countries

A Novel Strategy for Human Papillomavirus Detection and Genotyping with SybrGreen and Molecular Beacon Polymerase Chain Reaction

Human papillomaviruses (HPVs) play an important role in the pathogenesis of cervical cancer. For identification of the large number of different HPV types found in (pre)malignant lesions, a robust methodology is needed that combines general HPV detection with HPV genotyping. We have developed for formaldehyde-fixed samples a strategy that, in a homogenous, real-time fluorescence polymerase chain reaction (PCR)-based assay, accomplishes general HPV detection by SybrGreen reporting of HPV-DNA amplicons, and genotyping of seven prevalent HPV types (HPV-6, -11, -16, -18, -31, -33, -45) by real-time molecular beacon PCR. The false-positive rate of the HPV SybrGreen-PCR was 4%, making it well suited as a prescreening, general HPV detection technology. The type specificity of the seven selected HPV molecular beacons was 100% and double infections were readily identified. The multiplexing capacity of the HPV molecular beacon PCR was analyzed and up to three differently labeled molecular beacons could be used in one PCR reaction without observing cross talk. The inherent quantitation capacities of real-time fluorescence PCR allowed the determination of average HPV copy number per cell. We conclude that the HPV SybrGreen-PCR in combination with the HPV molecular beacon PCR provides a robust, sensitive, and quantitative general HPV detection and genotyping methodology