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This study aimed at resolving cellular genetic alterations in the process of in vitro immortalization of human keratinocytes by human papillomavirus (HPV) types 16 and 18. Four cell lines of primary human foreskin keratinocytes transfected with HPV 16 and HPV 18, respectively, were analysed during the transition from the mortal to immortal state. All cell lines showed strong telomerase activity at the immortal state, whereas no or only weak telomerase activity was detected in mortal precursor cells. This was consistent with telomere stabilization or restoration only observed in immortal cells, HPV physical state and copy number appeared constant during immortalization and HPV E6/E7 transcripts were present throughout. Immortalization was associated with clonal allele losses at 3p combined with either 11q or 18q or at 10p, dependent on the cell line. Moreover, a correlation was evident between the onset of telomerase activity and allele loss at 3p or 10p. All immortalized cells retained the capability to differentiate after growth in the presence of physiological calcium and serum. Moreover, one of the immortal cell lines displayed terminal differentiation after organotypic culturing on collagen rafts. The data suggest that (a) several pathways exist for HPV mediated immortalization that may involve genes residing at 3p, 10p, 11q and/or 18q; (b) 3p and 10p may encode genes involved in telomerase regulation; and (c) immortalization in vitro can be correlated with a spectrum of morphological changes varying from mild to severe dysplasia.

This study aimed at resolving cellular genetic alterations in the process of in vitro immortalization of human keratinocytes by human papillomavirus (HPV) types 16 and 18. Four cell lines of primary human foreskin keratinocytes transfected with HPV 16 and HPV 18, respectively, were analysed during the transition from the mortal to immortal state. All cell lines showed strong telomerase activity at the immortal state, whereas no or only weak telomerase activity was detected in mortal precursor cells. This was consistent with telomere stabilization or restoration only observed in immortal cells, HPV physical state and copy number appeared constant during immortalization and HPV E6/E7 transcripts were present throughout. Immortalization was associated with clonal allele losses at 3p combined with either 11q or 18q or at 10p, dependent on the cell line. Moreover, a correlation was evident between the onset of telomerase activity and allele loss at 3p or 10p. All immortalized cells retained the capability to differentiate after growth in the presence of physiological calcium and serum. Moreover, one of the immortal cell lines displayed terminal differentiation after organotypic culturing on collagen rafts. The data suggest that (a) several pathways exist for HPV mediated immortalization that may involve genes residing at 3p, 10p, 11q and/or 18q; (b) 3p and 10p may encode genes involved in telomerase regulation; and (c) immortalization in vitro can be correlated with a spectrum of morphological changes varying from mild to severe dysplasia.

A human papillomavirus (HPV) type 16 containing oral squamous cell carcinoma cell line 93VU147T at early passage was demonstrated to match its primary tumor with regard to HPV status and loss of heterozygosity at loci potentially involved in HPV-mediated carcinogenesis. DNA in situ hybridization of the cell line and the primary tumor revealed the presence of HPV 16 DNA clonally associated with the neoplastic cells. One- and two-dimensional Southern blot hybridization suggested HPV 16 to be integrated in the host genome at over hundred copies/cell. An identical restriction enzyme profile was observed for the tumor and the cell line. Viral DNA integration was confirmed by fluorescence in situ hybridization on metaphase spreads of the cell line, which revealed six stained loci comprising one at 15q14-15 and five at cytogenetically unidentifiable chromosomes. In addition, the tumor and the cell line displayed mRNA expression of the E6/E7 region encoding the viral oncoproteins, as determined by reverse transcription-PCR. Northern blot analysis of the cell line revealed three major and three minor transcripts harboring E67E7 sequences. Both the primary tumor and cell line showed loss of heterozygosity at the 11q22 (D11S35) and 18q21 (DCC) loci. These data support a role for HPV 16 in the development of a subset of oral cancers, presumably in concert with loss of function of tumor suppressor genes at 11q and 18q.

Cervical screening by high-risk HPV (hrHPV) testing requires additional risk stratification (triage), as most infections are transient and only a subset of hrHPV-positive women harbours clinically relevant disease. Molecular triage markers such as microRNAs (miRNAs) and DNA methylation markers are particularly promising, as they can be objectively tested directly on hrHPV-positive scrapes and cervicovaginal self-samples. Here, we evaluated the marker potential of 10 candidate miRNAs in 209 hrHPV-positive scrapes of women with underlying precancer (cervical intraepithelial neoplasia, grade 2–3 (CIN2-3)), cancer, or without disease (CIN0/1). A predictive miRNA classifier for CIN3 detection was built using logistic regression, which was compared to and combined with DNA methylation marker FAM19A4. Markers were correlated to histology parameters and hrHPV genotype. A miRNA classifier consisting of miR-149, miR-20a, and miR-93 achieved an area under the curve (AUC) of 0.834 for CIN3 detection, which was not significantly different to that of FAM19A4 methylation (AUC: 0.862, p = 0.591). Combining miRNA and methylation analysis demonstrated complementarity between both marker types (AUC: 0.939). While the miRNA classifier seemed more predictive for CIN2, FAM19A4 methylation was particularly high in HPV16-positive and histologically advanced CIN3, i.e. CIN3 with high lesion volume. The miRNA classifier, FAM19A4 methylation, and the miRNA/methylation combination were highest in cancer-associated scrapes. In conclusion, a panel of three miRNAs is discriminatory for CIN3 in hrHPV-positive scrapes and can complement DNA methylation analysis for the efficient detection of cervical disease. Combined analysis of the two marker types warrants further evaluation as triage strategy in hrHPV-based screening.

Cervical screening by high-risk HPV (hrHPV) testing requires additional risk stratification (triage), as most infections are transient and only a subset of hrHPV-positive women harbours clinically relevant disease. Molecular triage markers such as microRNAs (miRNAs) and DNA methylation markers are particularly promising, as they can be objectively tested directly on hrHPV-positive scrapes and cervicovaginal self-samples. Here, we evaluated the marker potential of 10 candidate miRNAs in 209 hrHPV-positive scrapes of women with underlying precancer (cervical intraepithelial neoplasia, grade 2–3 (CIN2-3)), cancer, or without disease (CIN0/1). A predictive miRNA classifier for CIN3 detection was built using logistic regression, which was compared to and combined with DNA methylation marker FAM19A4. Markers were correlated to histology parameters and hrHPV genotype. A miRNA classifier consisting of miR-149, miR-20a, and miR-93 achieved an area under the curve (AUC) of 0.834 for CIN3 detection, which was not significantly different to that of FAM19A4 methylation (AUC: 0.862, p = 0.591). Combining miRNA and methylation analysis demonstrated complementarity between both marker types (AUC: 0.939). While the miRNA classifier seemed more predictive for CIN2, FAM19A4 methylation was particularly high in HPV16-positive and histologically advanced CIN3, i.e. CIN3 with high lesion volume. The miRNA classifier, FAM19A4 methylation, and the miRNA/methylation combination were highest in cancer-associated scrapes. In conclusion, a panel of three miRNAs is discriminatory for CIN3 in hrHPV-positive scrapes and can complement DNA methylation analysis for the efficient detection of cervical disease. Combined analysis of the two marker types warrants further evaluation as triage strategy in hrHPV-based screening.

Cervical screening by high-risk HPV (hrHPV) testing requires additional risk stratification (triage), as most infections are transient and only a subset of hrHPV-positive women harbours clinically relevant disease. Molecular triage markers such as microRNAs (miRNAs) and DNA methylation markers are particularly promising, as they can be objectively tested directly on hrHPV-positive scrapes and cervicovaginal self-samples. Here, we evaluated the marker potential of 10 candidate miRNAs in 209 hrHPV-positive scrapes of women with underlying precancer (cervical intraepithelial neoplasia, grade 2–3 (CIN2-3)), cancer, or without disease (CIN0/1). A predictive miRNA classifier for CIN3 detection was built using logistic regression, which was compared to and combined with DNA methylation marker FAM19A4. Markers were correlated to histology parameters and hrHPV genotype. A miRNA classifier consisting of miR-149, miR-20a, and miR-93 achieved an area under the curve (AUC) of 0.834 for CIN3 detection, which was not significantly different to that of FAM19A4 methylation (AUC: 0.862, p = 0.591). Combining miRNA and methylation analysis demonstrated complementarity between both marker types (AUC: 0.939). While the miRNA classifier seemed more predictive for CIN2, FAM19A4 methylation was particularly high in HPV16-positive and histologically advanced CIN3, i.e. CIN3 with high lesion volume. The miRNA classifier, FAM19A4 methylation, and the miRNA/methylation combination were highest in cancer-associated scrapes. In conclusion, a panel of three miRNAs is discriminatory for CIN3 in hrHPV-positive scrapes and can complement DNA methylation analysis for the efficient detection of cervical disease. Combined analysis of the two marker types warrants further evaluation as triage strategy in hrHPV-based screening.