The telomere profile distinguishes two classes of genetically distinct cutaneous squamous cell carcinomas.

The incidence of skin cancer is increasing worldwide and cutaneous squamous cell carcinomas (SCCs) are associated with considerable morbidity and mortality, particularly in immunosuppressed individuals ('carcinomatous catastrophy'). Yet, molecular mechanisms are still insufficiently understood. Besides ultraviolet (UV)-indicative mutations, chromosomal aberrations are prominent. As telomeres are essential in preserving chromosome integrity, and telomere erosion as well as aberrant spatial telomere distribution contribute to genomic instability, we first established telomere length profiles across the whole tissue and identified normal skin (10/30) harboring discrete epidermal sites (stem cell territories) of evenly short telomeres. Precancerous actinic keratoses (AKs) (17) and SCCs (27) expressed two telomere phenotypes: (i) tissue-wide evenly short to intermediate and (ii) longer and tissue-wide heterogeneous telomere lengths, suggesting two modes of initiation, with one likely to originate in the epidermal stem cells. Although tumor histotype, location, patient gender or age failed to distinguish the two SCC telomere phenotypes, as did telomerase activity, we found a trend for a higher degree of aberrant p53 and cyclin D1 expression with long/heterogeneous telomeres. In addition, we established an association for the short/homogeneous telomeres with a simpler and the heterogeneous telomeres with a more complex karyotype correlating also with distinct chromosomal changes. SCCs (13) from renal transplant recipients displayed the same telomere dichotomy, suggesting that both telomere subtypes contribute to 'carcinomatous catastrophy' under immunosuppression by selecting for a common set (3, 9p and 17q) and subtype-specific aberrations (e.g., 6p gain, 13q loss). As a second mechanism of telomere-dependent genomic instability, we investigated changes in telomere distribution with its most severe form of telomeric aggregates (TAs). We identified a telomere length-independent but progression-dependent increase in cells with small telomere associations in AKs (17/17) and additional TAs in SCCs (24/32), basal cell carcinomas (30/31) and malignant melanomas (15/15), and provide evidence for a reactive oxygen species-dependent mechanism in this UV-induced telomere organization-dependent genomic instability.

High-resolution genomic profiling of human papillomavirus-associated vulval neoplasia.

BACKGROUND: The incidence of human papillomavirus-associated vulval neoplasia is increasing worldwide; yet the associated genetic changes remain poorly understood. METHODS: We have used single-nucleotide polymorphism microarray analysis to perform the first high-resolution investigation of genome-wide allelic imbalance in vulval neoplasia. Our sample series comprised 21 high-grade vulval intraepithelial neoplasia and 6 vulval squamous cell carcinomas, with paired non-lesional samples used to adjust for normal copy number variation. RESULTS: Overall the most common recurrent aberrations were gains at 1p and 20, with the most frequent deletions observed at 2q, 3p and 10. Copy-neutral loss of heterozygosity at 6p was a recurrent event in vulval intraepithelial neoplasia. The pattern of genetic alterations differed from the characteristic changes we previously identified in cutaneous squamous cell carcinomas. Vulval neoplasia samples did not exhibit gain at 5p, a frequent recurrent aberration in a series of cervical tumours analysed elsewhere using an identical protocol. CONCLUSION: This series of 27 vulval samples comprises the largest systematic genome-wide analysis of vulval neoplasia performed to date. Despite shared papillomavirus status and regional proximity, our data suggest that the frequency of certain genetic alterations may differ in vulval and cervical tumours.

Spontaneous keratinocyte cell lines representing early and advanced stages of malignant transformation of the epidermis.

A unique series of epidermal cell lines representing different stages of malignant transformation were spontaneously derived from a single adult immunosuppressed individual. Four keratinocyte lines (PM1-4) established from forehead skin are here compared with 4 squamous cell carcinoma (SCC) lines (MET1-4) derived respectively from a primary cutaneous tumour, two local recurrences and a distant metastasis of invasive SCC. Despite altered growth properties, the PM lines retained many features of normal keratinocytes including keratin phenotype, differentiation capacity and non-tumorigenicity in athymic mice. In contrast, from early passage, the MET lines displayed markedly reduced growth requirements, abnormal differentiation, aberrant K18 expression and tumorigenicity in athymic mice. The abnormal keratin profile of individual MET lines closely reflected the keratin phenotype of the tumour of origin. Although unusual HPV types were identified in the original tissue, there was no evidence of persistent virus within any cell line and it appears that HPV is not critical for maintenance of the immortal phenotype. The PM lines were distinctly different from invasive SCC lines and are likely to be useful for studies of mutations important early in neoplastic progression. The SCC series represent primary, recurrent and metastatic carcinoma. Availability of such a series from the same individual will facilitate genetic analysis of the malignant process.

The promoter of a novel human papillomavirus (HPV77) associated with skin cancer displays UV responsiveness, which is mediated through a consensus p53 binding sequence.

An aetiological role has been proposed for human papillomavirus (HPV) in skin carcinogenesis within the immunosuppressed patient population. To examine this possibility, we have focused on an HPV type that, to date, has been identified only in the cutaneous lesions of renal transplant recipients despite a high degree of sequence homology with other HPVs commonly found in warts in the general population. We report that the non-coding region of this virus, HPV type 77, contains a consensus binding site for the tumour suppressor protein p53, and we show by gel-retardation analysis that this sequence does indeed bind p53. Furthermore, using reporter gene assays, we demonstrate that HPV77 promoter activity is stimulated by UV radiation and that this response is mediated through the p53 binding site. This is the first report of a p53-dependent positive response element within a viral genome. Our results suggest a possible novel mechanism by which specific types of HPV might act as cofactors with UV radiation in cutaneous transformation.

Retention of low copy number human papillomavirus DNA in cultured cutaneous and mucosal wart keratinocytes.

Cultured wart keratinocytes have previously been described as having a limited proclivity to maintain episomal human papillomavirus (HPV) DNA. To investigate the nature of episome loss, and to determine keratinocyte-specific factors involved in it, we have examined a large series of anogenital and oral wart keratinocyte cultures, tracing episomal copy number with culture passage. We report that a higher proportion of oral wart keratinocytes maintain episomal HPV DNA to first passage (70% compared with 37% of anogenital wart cultures) when screened by slot blot hybridization. Furthermore, oral wart keratinocytes maintain episomal HPV copy through a greater number of passages (60% positive at passage 2 compared with 2% of anogenital wart cultures) with this technique. When anogenital cultures were examined at first passage for HPV infection by PCR with Southern blot hybridization of the product, a further 34% were found to be HPV-positive. To determine the mechanism of loss of episomal DNA from these cultures we examined the relative HPV copy number in cells which adhered to the culture vessel following passage and in those which did not adhere. Those which remained floating contained episomal HPV at high copy number whereas those which adhered were negative by slot blotting. The adherent cells, however, remained positive by PCR at subsequent passages until senescence. We conclude that a subpopulation of HPV-positive keratinocytes may be maintained in culture through serial passage until senescence.

Malignant transformation of cutaneous lesions in renal allograft patients: a role for human papillomavirus.

Renal allograft recipients are at greatly increased risk of developing squamous cell carcinomas. As these frequently arise adjacent to areas of multiple viral warts, we have investigated a possible role for human papillomavirus in malignant transformation within this population. We established, as primary cultures, keratinocytes from 24 lesions of varying degrees of squamous atypia from 9 patients. Ten of 14 cultures screened for the presence of episomal human papillomavirus DNA were positive using a mixed probe for cutaneous human papillomaviruses, although episomal copy was universally lost with continued passage. Three cultures, 2 of which were derived from malignant tissue and 1 from a benign lesion, were positive when screened with a probe for potentially oncogenic human papillomavirus DNAs 5 or 8. Both positive cultures of malignant origin exhibited extended lifespan and have been briefly characterized by morphology and growth requirements.

Human interleukin 3: effects on normal and leukemic cells.

The effects of recombinant human interleukin 3 (IL3) on normal bone marrow cells and human leukemic cells were studied. In clonal assays, IL3 supported the growth of all colony types including megakaryocytes. Erythroid colonies were formed in the presence of IL3 and erythropoietin, but not in the absence of erythropoietin. Replating experiments using blast cell colonies derived from a cell population enriched for progenitor cells by fluorescence-activated cell sorting with the monoclonal antibody 3C5, showed that IL3 supported the continued replating of colonies. The clonal proliferation of human bone marrow cells in response to IL3 was inhibited by tumor necrosis factor and by lymphotoxin, but not by interferon-gamma. In suspension cultures, IL3 supported the proliferation of mast cells. Human IL3 had no effect on the growth responses, morphology, cytochemistry, or clonogenicity of the human leukemic cell lines HL60, U-937, KG1a, and HEL. Transcripts for IL3 mRNA were not detectable in these cells, nor in the K562 cell line, implying that autocrine secretion of IL3 was not the mechanism by which these leukemias were maintained. Although cells derived from the bone marrow or peripheral blood of twenty patients with myeloproliferative disorders, myelodysplastic syndromes or acute myeloid leukemia frequently showed proliferative responses to IL3, mRNA transcripts for IL3 were not detected in these cells.