Biomedical subjects

Hanne Skomedal

Publications and source records attributed to Hanne Skomedal.

7 recordsLinked to original sources

PubMed2006

High-throughput detection of human papillomavirus-18 L1 gene methylation, a candidate biomarker for the progression of cervical neoplasia.

The L1 gene of human papillomavirus-18 (HPV-18) is consistently hypermethylated in cervical carcinomas, but frequently hypo- or unmethylated in exfoliated cells from asymptomatic patients. In precancerous lesions, L1 is sporadically hypermethylated, correlating with the severity of the neoplasia. In order to explore the potential of using L1 methylation as a workable biomarker for carcinogenic progression of HPV-18 infections in routinely taken samples, our aim was to develop methylation-detection techniques that were sensitive and rapid without being overly complex technically. Therein, we developed a methylation-specific PCR (MSP) through the design of primer sets that specifically amplify either methylated or unmethylated HPV-18 L1 DNA within bisulfite-modified sample DNA. Amplification of unmethylated and in vitro methylated HPV-18 DNA by MSP resulted in 2500 copies of either of the two L1 DNA species being detected, a satisfactory sensitivity considering that bisulfite treatment leads to the fragmentation of about 99% of sample DNA. The primers proved specific and did not generate false positive results at concentrations exceeding the lowest limit of detection by a factor of 400. DNA from carcinomas yielded PCR signals only with the methylation-specific primers, and not with primers specific for unmethylated L1 genes. The inverse result was obtained with DNA from precursor lesions that contained only hypomethylated DNA. High-grade precursor lesions and carcinomas that contained hyper- as well as hypomethylated L1 DNA yielded PCR signals with both primers. By developing a fluorescence based real-time PCR, we quantitatively analyzed samples with in vitro methylated and unmethylated L1 DNA, and could distinguish clinical samples with hyper- and hypomethylated DNA or mixtures of both DNAs. The methylation-specific and real-time PCR techniques permitted efficient HPV-18 L1 methylation analyses and open the door for larger-scale clinical studies where the utility of methylation status to predict the progression of HPV-18 infection and HPV-18 associated lesions is assessed.

Biomarkers↗

PubMed2006

Methylation of the human papillomavirus-18 L1 gene: a biomarker of neoplastic progression?

Epigenetic transcriptional regulation plays an important role in the life cycle of human papillomaviruses (HPVs) and the carcinogenic progression of anogenital HPV associated lesions. We performed a study designed to assess the methylation status of the HPV-18 genome, specifically of the late L1 gene, the adjacent long control region (LCR), and part of the E6 oncogene in cervical specimens with a range of pathological diagnoses. In asymptomatic infections and infections with precancerous (precursor) lesions, HPV-18 DNA was mostly unmethylated, with the exception of four samples where hypermethylation of L1 was detected. In contrast, L1 sequences were strongly methylated in all cervical carcinomas, while the LCR and E6 remained unmethylated. HeLa cells, derived from a cervical adenocarcinoma, contain chromosomally integrated HPV-18 genomes. We found that L1 is hypermethylated in these cells, while the LCR and E6 are unmethylated. Treatment of HeLa cells with the methylation inhibitor 5-Aza-2'-deoxycytidine (5-Aza-CdR) led to the expected reduction of L1 methylation. After removal of 5-Aza-CdR, L1 methylation resumed and exceeded pretreatment levels. Unexpectedly, the LCR and E6 also became methylated under these conditions, albeit at lower levels than L1. We hypothesize that L1 is preferentially methylated after integration of the HPV genome into the cellular DNA, possibly since linearization prohibits its normal transcription, while the enhancer and promoter may be protected from methylation by transcription factors. Since our data suggest that HPV-18 L1 methylation can only be detected in carcinomas, except in some few precancerous lesions and asymptomatic infections, L1 methylation may constitute a powerful molecular marker for detecting this important step of neoplastic progression.

Azacitidine↗

PubMed2006

Presence of E6 and E7 mRNA from human papillomavirus types 16, 18, 31, 33, and 45 in the majority of cervical carcinomas.

The oncogenic potential of the human papillomavirus (HPV) early genes E6 and E7 is well established and a source of interest with regard to HPV testing for cervical carcinoma. Here we present a study performed with 204 histologically confirmed invasive cervical squamous cell carcinomas (SCCs) in which we evaluated the HPV E6 and E7 mRNA detection assay PreTect HPV-Proofer for detection of high-risk HPV types 16, 18, 31, 33, and 45. For further evaluation, detection of E6 and E7 mRNA from HPV types 35, 52, and 58 by real-time multiplex nucleic acid sequence-based amplification was also included. For comparison and to assess the overall prevalence of various HPV types, samples were also tested for HPV DNA by both consensus and type-specific PCR, reverse line blotting, sequencing, and in situ hybridization. The overall prevalence of HPV was 97%. HPV E6 and E7 transcripts were detected in 188 of 204 (92%) biopsy specimens, of which 181 contained one of the following HPV types: 16, 18, 31, 33, or 45. Consensus PCR and type-specific PCR detected HPV in 187 of 204 and 188 of 204 (92%) specimens, respectively. In conclusion, this study verifies the presence of HPV E6 and E7 mRNA in SCCs and demonstrates that HPV infections among Norwegian women with SCCs are limited mainly to the five high-risk types, 16, 18, 31, 33, and 45. This, together with the fact that PreTect HPV-Proofer detects the HPV oncogenic transcripts, suggests that the assay is a valuable approach in the field of HPV detection in cervical carcinoma.

Carcinoma, Squamous Cell↗

PubMed2005

Predicting CIN2+ when detecting HPV mRNA and DNA by PreTect HPV-proofer and consensus PCR: A 2-year follow-up of women with ASCUS or LSIL Pap smear.

It has been suggested that human papillomavirus (HPV) testing improves follow-up of atypical cells of undetermined significance (ASCUS) and low-grade squamous intraepithelial lesion (LSIL) in cervical cancer screening programs. To evaluate the prognostic value of including HPV testing as an adjunct to cytology, we carried out a 2-year follow-up study of 77 women with ASCUS or LSIL Papanicolaou (Pap) smear in the Norwegian Cervical Cancer Screening Program (NCCSP) for detection of histological cervical intraepithelial neoplasia (CIN) 2+. The study includes a comparison between viral mRNA and DNA detection. PreTect HPV-Proofer was used for HPV E6/E7 mRNA detection from the 5 high-risk types 16, 18, 31, 33 and 45, and Gp5+/6+ consensus PCR was used for HPV DNA detection. Twice as many women were positive for HPV DNA (54.6%) than for HPV mRNA (23.4%). PreTect HPV-Proofer and consensus PCR had a sensitivity of 85.7% (95% confidence interval [CI] = 42.1-99.6) for detecting CIN2+ during follow-up. The specificity was significantly higher for PreTect HPV-Proofer, 84.9% (95% CI = 73.9-92.5), than for consensus PCR, 50.0% (95% CI = 37.4-62.6). PreTect HPV-Proofer positive women were 69.8 times (95% CI = 4.3-1137.3) more likely to be diagnosed with CIN2+ within 2 years than PreTect HPV-Proofer negative women. Consensus PCR-positive women were 5.7 times (95% CI = 0.6-52.0) more likely to be diagnosed with CIN2+ within 2 years than PCR-negative women. With equal sensitivity and higher specificity than consensus PCR, the PreTect HPV-Proofer might offer an improvement for the triage of women with ASCUS or LSIL Pap smear.

Adult↗

PubMed2005

Comparison of human papillomavirus messenger RNA and DNA detection: a cross-sectional study of 4,136 women >30 years of age with a 2-year follow-up of high-grade squamous intraepithelial lesion.

The purpose of this study was to compare the detection of human papillomavirus (HPV) DNA with detection of mRNA. The study included 4,136 women >30 years of age. E6/E7 mRNA expression from the carcinogenic HPV types 16, 18, 31, 33, and 45 was detected by the PreTect HPV-Proofer assay, whereas the presence of HPV DNA was detected by Gp5+/6+ consensus PCR followed by type-specific PCR. A total of 4.0% had an abnormal cytologic diagnosis, 3.0% were positive by PreTect HPV-Proofer, 4.4% by type-specific PCR, and 10.4% by consensus PCR. For detection of HPV in high-grade squamous intraepithelial lesion (HSIL), no significant difference was observed between PreTect HPV-Proofer and consensus PCR. For women with a cytologic normal, atypical squamous cell of uncertain significance, and low-grade SIL diagnosis, the detection rate of HPV was significantly higher by Gp5+/6+ consensus PCR (P < 0.005) than by PreTect HPV-Proofer. Histology confirmed 14 of 23 cytologic HSIL as cervical intraepithelial neoplasia grade >2. Of these women, PreTect HPV-Proofer and type-specific PCR detected 12, whereas consensus PCR detected 13. In conclusion, for HSIL, detection of E6/E7 transcripts from HPV types 16, 18, 31, 33, and 45 are present to the same degree as DNA detected by consensus PCR. Equally important, only a small proportion of the HPV DNA-positive women with a normal, atypical squamous cell of uncertain significance or low-grade SIL diagnosis had a detectable mRNA expression. HPV E6/E7 mRNA detection by PreTect HPV-Proofer represents a new promising test as an adjunct to cytology.

Adult↗

PubMed2004

Effect of the codon 72 polymorphism (c.215G>C, p.Arg72Pro) in combination with somatic sequence variants in the TP53 gene on survival in patients with advanced ovarian carcinoma.

This study was conducted to evaluate the frequency and prognostic impact of TP53 alterations stratified for the TP53 codon 72 polymorphism (c.215G>C, p.Arg72Pro) in a cohort of 109 patients with advanced ovarian carcinomas. TP53 sequence variants were observed in 80 of the 109 (73.4%) tumors and were significantly associated with grade of differentiation (P=0.001). A tendency towards higher frequency of sequence variants in tumors with higher FIGO stages was seen (P=0.05). The type of TP53 sequence variant (transition A:T>G:C vs. G:C>A:T at CpG dinucleotides, and transversion G:C>T:A) had significant correlation with patients' age (P=0.04) with more A:T>G:C in patients over 60 years old. No significant associations were found between frequency of sequence variants and age at diagnosis, histological type, size of residual tumor after primary surgery, or long-term survival. Analyses of the codon 72 polymorphism in tumor DNA gave a higher frequency of homozygosity/hemizygosity than expected from the population frequency, particularly for the Pro allele. Tumors homozygous or hemizygous for the Pro allele had significantly higher frequency of TP53 sequence variants, particularly of the nonmissense type (P=0.002), and patients with these types of alterations had significantly shorter survival (P=0.04). TP53 protein accumulation, determined by immunohistochemistry (IHC), was found in 67.9% (74 out of 109) of the tumors, was significantly more common among serous than nonserous ovarian carcinomas (P=0.008), and had a significant effect on progression-free survival (P=0.03). p63 (TP73L; formerly TP63) and p73 (TP73) protein accumulation detected by IHC was seen in 67.9 and 0% of the tumors, respectively. A significantly higher frequency of p63-positive cases was seen among serous tumors (P=0.008) and tended to increase with increasing FIGO stages (P=0.05), but had no significant effect on survival. No association between p63 protein accumulation and TP53 protein accumulation was seen.

Adult↗

PubMed2003

Gene expression patterns in ovarian carcinomas.

We used DNA microarrays to characterize the global gene expression patterns in surface epithelial cancers of the ovary. We identified groups of genes that distinguished the clear cell subtype from other ovarian carcinomas, grade I and II from grade III serous papillary carcinomas, and ovarian from breast carcinomas. Six clear cell carcinomas were distinguished from 36 other ovarian carcinomas (predominantly serous papillary) based on their gene expression patterns. The differences may yield insights into the worse prognosis and therapeutic resistance associated with clear cell carcinomas. A comparison of the gene expression patterns in the ovarian cancers to published data of gene expression in breast cancers revealed a large number of differentially expressed genes. We identified a group of 62 genes that correctly classified all 125 breast and ovarian cancer specimens. Among the best discriminators more highly expressed in the ovarian carcinomas were PAX8 (paired box gene 8), mesothelin, and ephrin-B1 (EFNB1). Although estrogen receptor was expressed in both the ovarian and breast cancers, genes that are coregulated with the estrogen receptor in breast cancers, including GATA-3, LIV-1, and X-box binding protein 1, did not show a similar pattern of coexpression in the ovarian cancers.

Adenocarcinoma↗