Induction of tetrasomy by human papillomavirus type 16 E7 protein is independent of pRb binding and disruption of differentiation.

We have demonstrated previously that high-risk human papillomaviruses (HPVs) induce tetrasomy in low-grade squamous intraepithelial lesions of the cervix. In this study we show that the E6 and E7 genes of high-risk HPV-16, but not those of low-risk HPV-6, are independently able to induce tetrasomy when constitutively expressed in proliferating monolayer cultures of primary human keratinocytes. Of seven HPV-16 E7 mutants analysed (H2P, Delta6-10, Delta21-24, C24G, S31G/S32G, A50S and S71I), five were severely impaired in their ability to induce tetrasomy in monolayer and raft culture. Only mutant C24G induced tetrasomy to levels comparable with wild-type E7 in monolayer and raft culture. This mutant shows strongly reduced binding to the retinoblastoma gene product pRb. The casein kinase II phosphorylation defective mutant S31G/S32G induced tetrasomy to levels comparable with wild-type E7 in raft culture, but not in monolayer culture, and induction of tetrasomy did not correlate with raft morphology. These results indicate that pRb protein binding is not required for HPV-16 E7 associated tetrasomy and that tetrasomy is not directly related to the ability of this protein to disrupt keratinocyte differentiation.

Tetrasomy is induced by human papillomavirus type 18 E7 gene expression in keratinocyte raft cultures.

We have demonstrated previously that oncogenic human papillomaviruses (HPVs) induce basal cell tetrasomy in low-grade squamous intraepithelial lesions of the cervix. To identify HPV genes and growth conditions involved in this process, we analyzed: (a) organotypic raft cultures of primary human keratinocytes transfected with whole HPV-18 genomes; and (b) organotypic raft cultures acutely infected with recombinant retroviruses expressing the HPV-18 E6, E7, or E6/E7 genes from the differentiation-dependent HPV-18 enhancer-promoter. Cultures were examined for HPV DNA by in situ hybridization and for karyotype by interphase cytogenetics. Tetrasomy occurred in the suprabasal strata of raft cultures expressing E7 and E6/E7 but not in those expressing E6 alone or in a control culture. These data indicate that suprabasal tetrasomy occurs in association with expression of the E7 gene alone. Basal cell tetrasomy was additionally observed in the raft culture transfected with whole HPV-18 genomes, consistent with observations in low-grade squamous intraepithelial lesions. The distribution of tetrasomic cells in these raft cultures may reflect the involvement of additional viral genes or possibly differences in the pattern of viral oncogene and host gene expression.

Loss of sequences on the short arm of chromosome 17 is a late event in squamous carcinoma of the cervix.

AIMS: To determine by fluorescence in situ hybridisation (FISH) whether deletion of D17S34, a subtelomeric probe for 17p, occurs in invasive squamous carcinoma of the cervix, and to determine the extent of such loss by analysis of the p53 and HER2/NEU genes. METHODS: Fourteen invasive squamous cell carcinomas of the cervix were investigated by FISH for D17S34, p53, and HER2/NEU. Dual hybridisation of each probe with the chromosome 17 alpha satellite (D17Z1) probe was performed on paraffin wax embedded sections, and the fluorescence ratios of the paired signals were determined. Broad spectrum human papillomavirus (HPV) typing by ISH and GP5+/6+ polymerase chain reaction was also performed. RESULTS: Twelve tumours were HPV positive, nine with HPV-16, and one each with types 18, 31, and 39. Loss of D17S34 was identified in four tumours, one of which was HPV negative. In one tumour, D17S34 loss was accompanied by loss of p53 only, suggesting that deletion was limited to the p arm. A second tumour showed simultaneous losses of all probes, indicative of whole chromosome 17 loss during tumour growth. The two remaining specimens showed loss of D17S34 only, diffuse in one, and localised within the tumour in the other. Aberrations of p53 or HER2/NEU were not seen independently of D17S34 loss, and loss did not correlate with HPV presence or type. CONCLUSIONS: These data show that D17S34 loss is prevalent, marking 28% of the invasive squamous carcinomas in this study. The observed intratumoral heterogeneity indicates that, at least in some cases, this loss occurs after invasion and is therefore a late event in the path of cervical carcinogenesis.

Loss of cytokeratin 14 expression is related to human papillomavirus type and lesion grade in squamous intraepithelial lesions of the cervix.

In a recent study of low-grade cervical squamous intraepithelial lesions (SILs), we reported that infection with both low- and high-risk human papillomaviruses (HPVs) upregulated cyclin A, B, E, and Ki67 expression in basal and suprabasal cells. In view of the intricate link between cell cycle exit, proliferation, and differentiation, we examined the morphologic distribution of cytokeratins 13 and 14 and involucrin expression in 49 low-grade SILs infected with HPV types 6, 11, 16, 18, 31, 33, 39, 42, 43, 44, 45, 51, 52, 56, 58, and 66; 2 lesions contained both low- and high-risk HPVs. The findings were compared with 30 high-grade SILs infected with HPV types 16, 31, 33, 51, 58, 66, and 67; 3 of these were infected with 2 different HPVs. In low-grade lesions, the differentiation markers were expressed normally, showing that differentiation proceeds despite upregulation of cell cycle--associated proteins. Loss of involucrin (3 of 33) and cytokeratin 13 (8 of 33) expression occurred only in the high-grade lesions and was therefore related to lesion grade. Loss of cytokeratin 14 expression was also significantly more frequent in high-grade than in low-grade lesions (19 of 33 v 12 of 51; P < .01). In addition, cytokeratin 14 expression was significantly less frequent in the intermediate and superficial layers of low-grade SILs infected with high-risk HPVs than in those infected with low-risk HPVs (3 of 27 v 14 of 24; P < .001). These findings are consistent with in vitro data and suggest that abnormalities of both cell cycle control and squamous differentiation are important in HPV-associated neoplastic transformation.

Verrucous carcinoma arising in pseudoepitheliomatous keratotic and micaceous balanitis, without evidence of human papillomavirus.

Pseudoepitheliomatous keratotic and micaceous balanitis (PKMB) is a condition which occurs on the glans penis of older men and may be associated with the development of a verrucous carcinoma. A role for human papillomavirus (HPV) in the aetiology of verrucous carcinoma has been implicated and several different HPV types have been found. We report a 74-year-old man who developed a verrucous carcinoma within an area of PKMB on the glans penis. Using a broad-spectrum polymerase chain reaction technique for identifying HPV, the epidermis of the area of PKMB and of the verrucous carcinoma were examined and no HPV DNA was identifiable. These results suggest that there is no part for HPV in the pathogenesis of PKMB or its transformation to verrucous carcinoma.

Evidence for keratinocyte immortalization in high-grade squamous intraepithelial lesions of the cervix infected with high-risk human papillomaviruses.

In this study, we demonstrate that expression of cyclin B protein is up-regulated and persists into the upper epithelial layers in parallel with cyclin A expression in high-grade squamous intraepithelial lesions (SIL) infected with human papillomaviruses 16, 31, 33, 51, 58, 66, and 67 (n = 33). In contrast, low-grade SIL infected with human papillomaviruses 16, 18, 31, 33, 39, 51, 52, 56, 58, and 66 (n = 27) show weaker cyclin B expression confined to basal and parabasal cells despite extension of cyclin A and Ki67 expression into superficial cells. Moreover, aneusomy is present in 20% of the high-grade lesions but in none of the low-grade lesions. The persistent expression of cyclin B in high-grade SIL, and the restriction of aneusomy to high-grade SIL suggest that there is cell cycle progression. In combination with in vitro studies, this provides evidence that high-grade SIL lesions have undergone immortalization.

Basal keratinocyte tetrasomy in low-grade squamous intra-epithelial lesions of the cervix is restricted to high and intermediate risk HPV infection but is not type-specific.

Human papillomavirus (HPV) infection appears to be an early event in cervical carcinogenesis with additional abnormalities being required for biological transformation. We have analysed 179 low-grade cervical squamous intra-epithelial lesions (SILs) and 15 normal cervices for the presence of HPV using both in situ hybridization and polymerase chain reaction (PCR). PCR was performed with GP5+/GP6+ primers followed by hybridization using probes for low (HPV 6, 11, 40, 42, 43, 44), intermediate (HPV 31, 33, 35, 39, 51, 52, 58, 59, 66 and 68) and high-risk HPVs (HPV 16, 18, 45 and 56). Interphase cytogenetic analysis using pericentromeric probes for chromosomes 1, 3, 4, 6, 10, 11, 17, 18 and X was also performed to identify numerical chromosomal abnormalities. Tetrasomy of all nine chromosomes was identified within basal keratinocytes, was restricted to epithelia infected with high risk (17 of 46) or intermediate risk (23 of 83) HPVs but was not HPV type-specific. Tetrasomy was not identified in any of the epithelia infected with low risk HPVs (n = 62). These numbers include multiple infection. These findings indicate that the induction of tetrasomy is a property restricted to high and intermediate-risk HPV types but that it is not type-specific. The factors governing which lesions will develop this abnormality are as yet unclear.

Disruption of cell cycle control by human papillomaviruses with special reference to cervical carcinoma.

Human papillomaviruses (HPVs) play a major role in neoplastic transformation of squamous epithelial cells. The viral genome is small in size and only encodes a limited number of proteins, so one of the major functions of the viral proteins is to modulate the function of key cellular proteins involved in cell cycle control and DNA replication. During this process important host cell cycle checkpoints are lost which may lead to the accumulation of genetic abnormalities and eventual malignant transformation. This review briefly describes the normal cell cycle and also the mechanisms by which HPVs interfere with cell cycle control both as part of their productive life cycle and in the process of neoplastic transformation.

Loss of retinoblastoma protein expression is frequent in small cell neuroendocrine carcinoma of the cervix and is unrelated to HPV type.

We have previously identified an inverse relationship between p53 and retinoblastoma protein (pRb) immunoreactivity in non-small cell carcinoma of the cervix. Because pRb is infrequently expressed in small cell carcinoma of the lung, we analyzed 25 small cell neuroendocrine carcinomas of the cervix to test the hypotheses that 1) lack of pRb expression is associated with the neuroendocrine phenotype in human papillomavirus (HPV)-associated cervical carcinoma and 2) the inverse relationship between p53 and pRb immunoreactivity also occurs in these tumors. HPV type was analyzed by PCR, HPV distribution by in situ hybridization and expression of p53 and pRb by immunohistochemistry. All of the tumors contained HPV sequences, with 13 tumors HPV 16 positive, 11 HPV 18 positive, and 1 HPV 45 positive. In situ hybridization showed large intranuclear dot-like signals in all positive tumors, suggesting viral integration. No multiple infections were identified. Expression of retinoblastoma protein was not detectable in 23 tumors (92%), the remaining two showing only weak, focal expression. Expression of p53 protein was variable in distribution and intensity. It did not correlate with HPV type, and there was no relationship with pRb immunoreactivity. These data indicate that, although there is no reciprocal relationship between p53 and pRb immunoreactivity in these tumors, retinoblastoma protein is infrequently expressed in HPV-containing small cell neuroendocrine carcinoma of the cervix, irrespective of infecting HPV type. This is consistent with the reported findings in small cell carcinoma of the lung and suggests that the small cell neuroendocrine phenotype may be related to the abrogation of retinoblastoma protein function.

p53 codon 72 ARG/PRO polymorphism is not related to HPV type or lesion grade in low- and high-grade squamous intra-epithelial lesions and invasive squamous carcinoma of the cervix.

A polymorphism at codon 72 of the p53 gene, resulting in either an arginine or a proline residue in the protein, has been reported to affect the susceptibility of p53 to human papillomavirus (HPV) E6-mediated degradation in cultured cells. However, the relevance of this polymorphism to naturally occurring HPV infection is unclear. Therefore, we analysed its relationship to infecting HPV type and lesion grade in a series of low- and high-grade squamous intra-epithelial lesions (SILs) and invasive carcinoma of the cervix. DNA extracted from morphologically characterised, paraffin-embedded tissues (30 normal cervices, 118 low-grade SILs, 118 high-grade SILs and 43 invasive carcinomas) was examined for the presence and type of HPV DNA, and the p53 genotype was identified by both allele-specific PCR and PCR-restriction fragment length polymorphism. There was no significant relationship between the frequency of p53 genotypes and either HPV type or lesion grade. Our data do not support the hypothesis that this p53 polymorphism is involved in the development of high-grade squamous cervical disease in this population.

Discrimination of human papillomavirus types in low and high grade cervical squamous neoplasia by in situ hybridization.

We have previously demonstrated that cross-hybridization between probe and target occurs in the analysis of human papillomavirus (HPV) 6 and 11, and HPV 16, 31 and 33 infection by in situ hybridization (ISH) in archival tissue biopsies. In this study, 50 low grade and 50 high grade cervical lesions were analyzed using HPV 6/11, 16, 18, 31 and 33 probes to determine the typing accuracy of high sensitivity ISH for a wide range of HPV types. The sensitivity of both ISH and PCR was 92% in low grade lesions and 90% and 96%, respectively, in high grade lesions, with an overall concordance of 97%. The typing accuracy of ISH was only 43% in low grade lesions but 93% in high grade lesions. This was due largely to cross-hybridization of the probes used with other HPV types (HPV 39, 42, 43, 44, 45, 51, 52, 56, 58, and 66). Although analysis of ISH patterns could narrow down these other HPV types, precise identification was not possible by this means. These data suggest that when high sensitivity ISH methods are used, particularly in low grade lesions, HPV typing by ISH should be supplemented by independent determination of HPV type by PCR.

Differential cell cycle regulation by low- and high-risk human papillomaviruses in low-grade squamous intraepithelial lesions of the cervix.

In this study, we have demonstrated the overexpression of cyclin D1 protein in 24 (92%) of 26 low-grade squamous intraepithelial lesions infected with low-risk human papillomaviruses (HPV 6, 11, 42, 43, and 44) and the absence of cyclin D1 expression in 25 (87%) of 29 lesions infected with high-risk HPVs (HPV 16, 18, 31, 33, 39, 45, 51, 52, 56, 58, and 66). The expression of cyclins E, A, and B proteins was increased in both low-risk and high-risk HPV infections. Tetrasomy of chromosomes 1, 3, 11, 17, 18, and X was present in nine lesions, all of which were infected with high-risk HPVs, but was not related to the pattern of cyclin expression. These data provide in vivo evidence that low- and high-risk HPV types alter cell cycle control by different mechanisms and that cell cycle checkpoint abnormalities are induced by high-risk, but not low-risk, HPV infection.

Molecular events in uterine cervical cancer.

OBJECTIVE: To review the literature regarding the molecular events which occur in the development of uterine cervical cancer, with particular reference to human papillomavirus (HPV) infection. METHODOLOGY: Bibliographic searches of Medline and the ISI citation databases using appropriate keywords, including the following: papillomavirus, cervix, pathology, cyclin, chromosome, heterozygosity, telomerase, smoking, hormones, HLA, immune response, HIV, HSV, EBV. CONCLUSIONS: It has become clear that most cervical neoplasia, whether intraepithelial or invasive, is attributable in part to HPV infection. However, HPV infection alone is not sufficient, and, in a small proportion of cases, may not be necessary for malignant transformation. There is increasing evidence that HPV gene products interfere with cell cycle control leading to secondary accumulation of small and large scale genetic abnormalities. This may explain the association of viral persistence with lesion progression but, in many patients, secondary factors, such as smoking and immune response, are clearly important. However, the mechanisms involved in the interaction between HPV and host factors are poorly understood.

Interphase cytogenetic evidence for distinct genetic pathways in the development of squamous neoplasia of the uterine cervix.

Human papillomavirus (HPV) infection has been implicated as an etiologic factor in most cervical cancers. However, additional genetic alterations are thought to be required for the development of a carcinogenic genotype. In the present study, interphase cytogenetics utilizing pericentromeric probes specific for chromosomes 1, 3, 11, 17, 18, and X was performed on paraffin-embedded tissue sections from 25 high-grade squamous intraepithelial lesions (SILs) and 25 invasive squamous cell carcinomas (ISCCs) of the cervix. HPV infection was determined by both in situ hybridization and broad-spectrum GP5+/GP6+ PCR. HPV was identified in all high-grade SILs (HPV 16, n = 16; 18, n = 2; 26, n = 1; 31, n = 4; 45, n = 1; 66, n = 1) and 23 (92%) ISCCs (HPV 16, n = 19; 18, n = 2; 31, n = 1; 39, n = 1). Aneusomy was identified in 11 (44%) high-grade SILs and 18 (72%) ISCCs. In 18 (62%) of these, relative under-representation of chromosomes 3, 11, 17, and/or 18 was identified (8 high-grade SILs and 10 ISCCs). Tetrasomy of all six chromosomes was present in two high-grade SILs but no ISCCs. Twelve (48%) high-grade SILs and seven (28%) ISCCs were disomic with all six chromosome probes, and there was no relationship between HPV presence or type and chromosome pattern. The presence of distinct patterns of numerical chromosome abnormality in these lesions suggests that progression to high-grade SIL or invasive carcinoma can occur by more than one genetic pathway. The lack of correlation between chromosome pattern and HPV type indicates that these pathways are not HPV type-specific. Whether these patterns reflect differences in early gene expression, possibly related to viral integration, or differences in the biologic properties of HPV type variants remains to be established.

Basal cell tetrasomy in low-grade cervical squamous intraepithelial lesions infected with high-risk human papillomaviruses.

We have analyzed 60 low-grade cervical squamous intraepithelial lesions for low- and high-risk human papillomaviruses (HPVs) and for numerical abnormalities of chromosomes 1, 3, 11, 17, and 18 and the X chromosome. Eleven of 33 lesions infected with high-risk HPVs (HPV 16, 18, 30, 31, 33, 35, 39, 45, 51, 52, 56, 58, and 66) but none of 24 lesions infected with low-risk HPVs (HPV 6, 11, 42, 43, and 44) and none of 15 normal cervices showed basal cell tetrasomy of all six chromosomes in the HPV-infected areas. These changes were not HPV type specific and were not present in all lesions infected with the same HPV type. The presence of basal cell tetrasomy in lesions infected with high- but not low-risk HPVs suggests that induction of chromosome instability may be one mechanism underlying the biological differences between these viral types.

Interphase cytogenetic demonstration of chromosome 9 loss in thick melanomas.

It has been postulated that deletion of genes on chromosome 9 is important in the development of malignant melanoma. In this study, we have investigated this hypothesis by analysing the numerical complement of chromosomes 9, 17 and X by interphase cytogenetics using peri-centromeric repeat probes on paraffin sections from 15 thick melanomas. Three cases showed no relative loss or gain of chromosomes. Two cases showed gain of chromosome 17, and one case loss of chromosome 17 relative to chromosomes 9 and X. Relative chromosome 9 loss was identified in 9 cases (60%). Two of these were monosomic for chromosome 9 with a normal complement of chromosomes 17 and X and six were tetrasomic for chromosome 17 with duplication of chromosome X: chromosome 9 was disomic in five of these cases and trisomic in one. The final case showed loss of both chromosomes 9 and 17 relative to X. The chromosome patterns obtained imply that loss of chromosome 9 frequently takes place before tetraploidisation. This is in keeping with the hypothesis that loss of chromosome 9 is not a late event in melanocyte transformation. Extension of these studies to thin melanomas, in situ melanomas and dysplastic naevi will refine further the point at which these changes occur.

Interphase karyotypic analysis of chromosomes 11, 17 and X in invasive squamous-cell carcinoma of the cervix: morphological correlation with HPV infection.

Human papillomavirus (HPV) infection has been widely implicated in cervical carcinogenesis, but it appears to be an early event, with other genetic abnormalities being required for biological transformation. In this study, interphase cytogenetic analysis of numerical abnormalities of chromosomes 11, 17 and X was performed on paraffin-embedded tissue sections from 25 invasive squamous-cell carcinomas of the cervix and compared with both histopathological features and the morphological distribution of HPV sequences as determined by in situ hybridisation. Numerical differences between chromosomes were identified in 76% of cases, with underrepresentation of chromosomes 11 and/or 17 relative to X in 64% of the total; 22 of 25 cases were HPV-positive, containing either HPV 16, 18 or 31. There was no relationship between the distribution of viral sequences and chromosomal pattern, suggesting that HPV infection precedes karyotypic changes. Our findings suggest that relative reduction in number of chromosomes 11 and 17 is important in the development of invasive cervical neoplasia and are consistent with the putative presence of relevant tumour-suppressor genes on these chromosomes.

Detection of interleukin-8 mRNA and protein in human colorectal carcinoma cells.

Interleukin-8 (IL-8) is a member of the chemokine family of pro-inflammatory chemotactic cytokines and is secreted by some human colorectal carcinoma cell lines. We have used in situ hybridisation and immunohistochemistry to determine whether IL-8 mRNA and protein, respectively, are produced by human colorectal carcinoma cells in vivo. IL-8 mRNA was detected within the cytoplasm of tumour cells in all nine samples tested, including that of a tumour which had metastasised to a lymph node. Non-involved colonic mucosa within the same tissue blocks showed much weaker labelling. IL-8 protein was detected in 74% (23/31) of tumour samples and was mainly localised to the tumour cell cytoplasm. In 30% of cases, staining was heterogeneous, with between 1 and 30% of cells being positive. In some tumour cells, IL-8 showed a perinuclear distribution resembling that found by in situ hybridisation. Some infiltrating leucocytes, endothelial cells and fibroblast-like cells within the tumour sections were also positive for IL-8 mRNA and protein. The possibilities that colorectal tumours produce IL-8 to aid invasion and/or metastasis or as a tumour growth factor are discussed.

Assessment of intra-tumoral karyotypic heterogeneity by interphase cytogenetics in paraffin wax sections.

Aim-To analyse the effect of sectioning on the assessment of karyotypic heterogeneity by interphase cytogenetics in paraffin wax embedded normal squamous epithelium and to apply the principles derived to invasive cervical carcinoma.Methods-Normal male (n = 5) and female (n = 5) squamous epithelia were hybridised with peri-centromeric repeat probes specific for chromosomes X (DXZ1) and 17 (D17Z1) individually and in combination to assess the effect of sectioning on mono-, di-, tri-, and tetrasomic populations. Section thickness, interobserver variation and variation between different areas of the epithelium were evaluated. Invasive squamous carcinomas of the cervix (n = 5) were then hybridised with the DXZ1 probe and intratumoral heterogeneity was assessed by comparison of signal distributions obtained from different areas.Results-The optimum section thickness for the assessment of normal epithelium was 6 mum. Variation in the expected signal number in the range 1-4 did not introduce artefactual heterogeneity at this section thickness. The sensitivity of this approach for the detection of minor subpopulations was calculated to be 13-16%, 17-18% and 10-11% for mono-, tri- and tetrasomic populations, respectively. Karyotypic heterogeneity was detected in two of the five tumours and, in one case where the populations where clustered morphologically, a minor population representing 18% was identified.Conclusions-Interphase cytogenetic analysis of sections from paraffin wax embedded material can be used for the detection of minor subpopulations in tumours. This approach will be of particular value in the assessment of the relation between human papillomavirus infection and tumour karyotype and in the analysis of intraepithelial neoplasia.