Irene Kraus

Type-Dependent Integration Frequency of Human Papillomavirus Genomes in Cervical Lesions

Chromosomal integration of high-risk human papillomavirus (HR-HPV) genomes is believed to represent a significant event in the pathogenesis of cervical cancer associated with progression from preneoplastic lesions to invasive carcinomas. This hypothesis is based on experimental data suggesting that integration-dependent disruption of HR-HPV E2 gene functions is important to achieve neoplastic transformation and on clinical data gathered by analyzing lesions induced by human papillomavirus (HPV) 16 and 18 that revealed integrated viral genome copies in the vast majority of cervical cancer cells. However, a substantial fraction of cervical cancers is associated with other HR-HPV types for which virtually no data concerning their integration status have been reported so far. Here, we compared integration frequencies of the five most common oncogenic HPV types (HPV16, 18, 31, 33, and 45) in a series of 835 cervical samples using a specific mRNA-based PCR assay (Amplification of Papillomavirus Oncogene Transcripts). Most precancerous lesions displayed exclusively episomal viral genomes, whereas 62% of the carcinomas had integrated viral genomes. However, the frequency of integrated HR-HPV genomes showed marked differences for individual HR-HPV types. HPV16, 18, and 45 were found substantially more often in the integrated state compared with HPV types 31 and 33. The analysis of the median age of patients with high-grade precancerous lesions and invasive cancers suggests that precancers induced by HPV types 18, 16, and 45 progress to invasive cervical cancer in substantially less time compared with precancers induced by HPV types 31 and 33. These findings suggest that integration of oncogenic HPV genomes in cervical lesions is a consequence rather than the cause of chromosomal instability induced by deregulated HR-HPV E6-E7 oncogene expression. Distinct HR-HPV types apparently provoke chromosomal instability in their host cells to a different extent than is reflected by their integration frequencies in advanced lesions and the time required for CIN 3 lesions to progress to invasive cancer.

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.

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.

Presence of E6 and E7 mRNA from Human Papillomavirus Types 16, 18, 31, 33, and 45 in the Majority of Cervical Carcinomas

ABSTRACT 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.

The N-Terminal Domain of Thyroid Hormone Receptor-α Is Required for Its Biological Activities

Thyroid hormone (T3) receptors (T3Rs) are ligand-modulated transcription factors that belong to the nuclear receptor superfamily. Whereas the well-conserved DNA-binding domain and the relatively well-conserved ligand-binding domain in T3Rs have been characterized in detail, limited information is available on the contribution of the variable N terminus to the transcriptional properties of T3Rs. To gain greater insight into the function of the N terminus, we generated a deletion mutant of T3Ralpha, T3Ralpha-deltaN1, that lacks amino acids 7-45 and assessed the effect of this deletion on all known transcriptional activities of T3Ralpha. Despite the fact that T3Ralpha-deltaN1 was expressed and bound T3 with an affinity similar to that of wildtype T3Ralpha, all of its common transcriptional activities were lost. That is, T3Ralpha-deltaN1 did not activate transcription from a positive or negative T3 response element, and it could not interfere with AP-1 transcriptional activity. Surprisingly, T3Ralpha-deltaN1 lost its ability to bind DNA, which can account for its deficiencies as a transcriptional activator. In contrast, the ability of T3Ralpha-deltaN1 to interact with putative coactivators or corepressors was not significantly altered from that of wildtype T3Ralpha. However, overall folding of T3Ralpha-deltaN1 was altered, as indicated by differential sensitivity to limited protease digestion. These data document that the N terminus of T3Ralpha, albeit relatively short and representing a variable and unconserved region when compared with other nuclear receptors, has a critical role in proper folding of the DNA-binding domain and is required for the biological activities of full-length T3Ralpha.

Human papillomavirus DNA and e6/e7 mRNA status in relation to survival of patients treated for cervical squamous cell carcinoma.

The prognostic significance of human papillomavirus (HPV) DNA and E6/E7 mRNA, the presence of specific types, and the physical state of HPV DNA, were studied in 202 cervical squamous cell carcinomas. Absence or non-detectable levels of high-risk (types 16, 18, 31, 33, 35, 45, 52 and 58) E6/E7 mRNA, using the real-time nucleic acid sequence based amplification (NASBA) assay, and absence of HPV high-risk/HPV 6, 26, 66, 69, 73 (all methods collectively) were associated with poor overall survival in univariate analysis (P = 0.04 and P = 0.03, respectively) and had independent prognostic value in multivariate analysis (P = 0.01 and P = 0.03, respectively) including FIGO stage and age. Based on the individual results of type-specific PCR and in situ hybridization (ISH), the presence of HPV DNA was not found to be a prognostic factor. Likewise, concerning the presence of specific HPV types and the HPV integration status (determined by ISH), no prognostic significance was found. Mutation analyses of the TP53 gene revealed mutations in 3 of the 6 HPV negative samples (50%).