Search

Aims: To describe the clinicopathological features of florid peritoneal mesothelial hyperplasia associated with gynaecological disease., Methods: Review of a series of 44 cases where there was significant peritoneal mesothelial proliferation, usually on the surface of the ovary, which resulted in diagnostic problems, often including a consideration of malignancy., Results: Florid mesothelial proliferation was associated with a variety of benign and malignant lesions, most commonly endometriosis. The most characteristic morphological appearance was that of small bland tubules and nests and cords of cells often embedded in fibrous tissue, sometimes with a linear arrangement. In a minority of cases, there were closely packed small glands and papillae resulting in mimicry of a serous proliferation; psammoma bodies were present in one such case. In several cases, the groups of mesothelial cells either exhibited true lymphovascular invasion or were surrounded by spaces closely simulating lymphovascular invasion. Although most cases were appreciated to represent a reactive mesothelial proliferation, several were referred with a presumed or possible diagnosis of mesothelioma, low-grade serous carcinoma, adenocarcinoma, adenomatoid tumour or Sertoli cell tumour. Positive staining with calretinin and negative staining with Ber-EP4 often helped to identify the cells as mesothelial rather than epithelial., Conclusions: Florid peritoneal mesothelial hyperplasia can occur in association with a variety of gynaecological disorders. Recognition of this phenomenon and appreciation that it can produce worrisome histological patterns, particularly when associated with endometriosis, is key to reaching the correct diagnosis.

Gynaecological tumours exemplify many of the molecular paradigms of carcinogenesis. The clinical value of many of the molecular abnormalities present is now being tested and it is likely that the identification of at least some of these will become routine in the near future. This may help to refine diagnosis and guide treatment-for example, therapeutic vaccination for human papillomavirus related disease.

Aim: To determine the prevalence and significance of human papillomaviral types in conjunctival pterygia., Methods: Polymerase chain reaction technology was used to identify the presence of human papillomavirus (HPV) in 10 formalin fixed paraffin embedded pterygia samples. 10 conjunctival papillomas were used as positive controls. 20 conjunctival samples, 10 with primary acquired melanosis and 10 with malignant melanoma, were used as negative controls. Sample subgroups were of equal sex, race, and age distribution to eliminate bias. All samples were further analysed (for 21 HPV types) using dot-blot hybridisation techniques., Results: HPV was identified in 90% of the conjunctival papillomas, 50% of the pterygia samples, but no HPV was detected in the negative control group. Two pterygia showed type 6, two type 11, and one type 16. These three HPV types were also detected in papillomas., Conclusion: These results suggest that HPV may be involved in the pathogenesis of pterygia and that broadly the same HPV types are found in pterygia and in papillomas. Persistent conjunctival HPV may possibly play a part in the recurrence of pterygia post excision but further larger studies are required to elucidate this hypothesis.

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.

Aim: To test the hypothesis that, because the human papillomavirus (HPV) E2 protein represses viral early gene transcription, E2 gene sequence variation or disruption could play a part in the induction of the numerical chromosome abnormalities that have been described in squamous cervical lesions., Methods: The integrity and sequence of the E2 gene from 11 cervical intraepithelial neoplasia (CIN) grade 3 lesions and 14 invasive squamous cell carcinomas, all of which contained HPV-16, were analysed by the polymerase chain reaction (PCR). The E2 gene was amplified in three overlapping fragments and PCR products sequenced directly. Chromosome abnormalities were identified by interphase cytogenetics using chromosome specific probes for chromosomes 1, 3, 11, 17, 18, and X., Results: E2 gene disruption was present in significantly more invasive carcinomas (eight of 14) than CIN 3 lesions (one of 11) (p = 0.03). No association was found between E2 disruption and the presence of a numerical chromosome abnormality. The E2 gene from the non-disrupted isolates was sequenced and wild-type (n = 5) and variant (n = 11) sequences identified. Variant sequences belonged to European and African classes and contained from one to 15 amino acid substitutions. Although numerical chromosome abnormalities were significantly more frequent in invasive squamous cell carcinoma than CIN 3 (p = 0.04), there was no significant relation between the presence of sequence variation and either histological diagnosis or chromosome abnormality., Conclusions: These data do not support the hypothesis that E2 gene disruption or variation is important in the induction of chromosome imbalance in these lesions. However, there is a relation between E2 gene disruption and the presence of invasive disease.

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.

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.

Aim: To assess the diagnostic performance of human papillomavirus (HPV) analysis in predicting cervical intraepithelial neoplasia (CIN) grades 2 and 3 in patients with persistent low grade cervical cytological abnormalities., Methods: Cervical smears from 167 women referred for colposcopy with persistent borderline, wart virus or mildly dyskaryotic changes on cervical screening were analysed by Papanicolaou staining, non-isotopic in situ hybridisation and generic and type specific polymerase chain reaction (PCR) amplification of HPV sequences. Follow up was by cytological and, where appropriate, histological analysis., Results: CIN grade 2 or 3 was identified in 46 patients after a median follow up of 27 months. HPV positivity by both techniques was associated with high grade CIN and with age less than 30 years (median age 33 years). Non-isotopic in situ hybridisation was more predictive but less sensitive than either generic or type specific PCR, but prediction was greater using either molecular technique in women over 30 years of age., Conclusions: Although the degree of prediction found is of only limited clinical value, the strong association of HPV positivity with both high grade CIN and patient age suggests that further studies of HPV testing in this patient group are warranted.

Aim: To investigate the association of human immunodeficiency virus (HIV) infection and carcinoma of the conjunctiva in Africa, and the role of human papilloma virus type 16 (HPV-16)., Methods: Patients in Uganda and Malawi presenting to eye clinics with lesions suspicious of carcinoma were studied. Pathological confirmation of eye lesions was sought. HIV testing of patients who were biopsied and, in Uganda, of matched case control subjects was carried out as was testing of a sample of fixed biopsies for HPV-16 by polymerase chain reaction (PCR). The HIV-1 serology, histopathology of conjunctival biopsies (conjunctival intraepithelial neoplasia (CIN), invasive carcinoma, other lesions), and prevalence of HPV-16 infection were determined., Results: Of Ugandan patients, 27/38 (71%) with carcinoma (27 invasive carcinoma, 11, CIN) were HIV positive compared with 12/76 (16%) of controls (odds ratio 13, 95% confidence interval 5-38). The calculated population aetiological fraction of carcinoma associated with HIV was 66%. Of 32 Malawian patients (20 invasive carcinoma, 12 CIN), 25/29 tested (86%) were HIV positive. HPV-16 infection was found in 7/20 (35%) of carcinoma samples, 0/9 pingueculae, and 2/6 conjunctivitis samples., Conclusions: HIV infection is strongly associated with an apparent increase in the incidence of conjunctival carcinoma in Africa. While ultraviolet light is probably the prime risk factor and HPV-16 is implicated in a proportion of cases, the interactions of ultraviolet light, HIV, HPVs, and other factors are unclear in the pathogenesis of carcinoma. The disease represents another model of multifactorial epithelial carcinogenesis.

AIMS--To assess the relative diagnostic performance of the polymerase chain reaction (PCR) and non-isotopic in situ hybridisation (NISH) and to correlate these data with cytopathological assessment. METHODS--Paired analysis of human papillomavirus (HPV) detection was performed by PCR and NISH on exfoliated cervical cells from 122 women attending a routine gynaecological examination. PCR amplification followed by generic and HPV type specific hybridisation was compared with NISH on a parallel cervical smear. RESULTS--Overall, 32 cases were positive by NISH and 61 positive by PCR. Of the 105 cases in which both PCR and NISH were interpretable, 76 (26%) were normal smears, 20 of which were HPV positive by NISH and 37 (49%) by PCR. Of 17 borderline smears, two were NISH positive and 12 PCR positive. Eight of nine smears containing koilocytes were positive by NISH and seven by PCR. Of three dyskaryotic smears, none were NISH and two were PCR positive. The concordance of NISH and PCR in these samples was 57%. To assess sampling error, NISH and PCR were performed on an additional 50 cases using aliquots from the same sample. This increased the concordance between assays to 74%. Filter hybridisation of PCR products with the cocktail of probes used in NISH (under low and high stringency conditions) demonstrated that several cases of NISH positivity could be accounted for by cross-hybridisation to HPV types identified by PCR but not present in the NISH probe cocktail. CONCLUSIONS--Sampling error and potential cross-hybridisation of probe and target should be considered in interpretation of these techniques. PCR is more sensitive because it provides for the amplification of target DNA sequences. In addition, the PCR assay utilised in this study detects a wider range of HPV types than are contained in the cocktails used for NISH. However, PCR assays detect viral DNA present both within cells and in cervical fluid whereas NISH permits morphological localisation.

Aims: To investigate the role of human papillomavirus (HPV) in the association between HLA DQw3 and squamous cell cancer of the cervix (SCCC)., Methods: Tissue from 194 cervical samples, ranging from normal, through cervical intraepithelial neoplasia, to SCCC, were typed for HPV by amplification of the L1 gene using degenerate consensus primers, followed by oligonucleotide probing. HLA DQw3 typing was undertaken in the same samples using a new PCR amplification system using primers common to all DQ loci, followed by restriction digestion with Mlu 1 to differentiate HLA DQw3 types--null, heterozygous, and homozygous. The data were analysed using chi 2 analysis and by calculating relative risks with the 95% confidence interval., Results: Samples (n = 188) were successfully typed for HPV and 177 were typed for HLA DQw3. There was a nonsignificant rise in the prevalence of HLA DQw3 in SCCC (64.3%) compared with the group with normal histology (53.2%). Analysis of the prevalence of HLA DQw3 on the basis of HPV infection rather than histology showed that 63 of 95 (66.3%) of the HPV positive samples contained HLA DQw3 alleles, compared with 39 of 78 (50.0%) of the HPV negative samples (chi 2 4.06; p < 0.05)., Conclusions: There was a significant association between HLA DQw3 and cervical HPV infection. This may be because people with HLA DQw3 are less able to mount an effective immune response to HPV, which predisposes them to the development of SCCC.

Aims: To determine the relative diagnostic performance of non-isotopic in situ hybridisation (NISH) and a dot-blot assay for detecting human papillomavirus (HPV) on exfoliated cervical cells; and to correlate the results with cytopathological assessment., Methods: Cervical smears and cytological samples were obtained from 122 patients during the same clinical examination and the presence of HPV sequences determined by NISH and dot-blot analysis, respectively., Results: Dot-blot analysis gave an autoradiographic signal in 15 of 121 (12.4%) cases, while NISH detected viral genomes in 38 of 114 (33.3%) cases. Even in the presence of koilocytosis, where vegetative replication of the virus occurs, NISH was positive in over twice as many cases as dot-blot analysis (NISH 90%, dot-blot 40%), while in smears within normal cytological limits, where the viral copy number is likely to be considerably lower, the differences were more striking (NISH 31%, dot-blot 5%)., Conclusions: These data show that NISH on cytological smears is more sensitive than a standardised dot-blot hybridisation assay for detecting HPV infection in cytological material and is therefore a more appropriate screening tool.

Aim: To develop a methodology for direct detection of high risk human papillomavirus (HPV) infection in routine cervical smears by non-isotopic in situ hybridisation (NISH) which can be compared with cytopathological assessment of the same cells., Methods: The methodology was established using cultured cells and routine cervical smears hybridised with digoxigenin labelled probes for HPV, 16, 18, 31, and 33. The technique was applied to the analysis of 53 patients from a sexually transmitted disease clinic., Results: The optimal sensitivity achieved for single HPV detection in cultured cells was 1-2 copies of HPV 16 per cell and that for detection of a cocktail of HPV types in routine cervical smears was 2.5-12 copies per cell. Of parallel smears taken from patients with a normal Papinacolau-stained smear 33.3% (24) contained a HPV 16, 18, 31, and 33 signal indicating an occult HPV infection. The prevalence of these HPV types was similar in women in whom a cytopathological diagnosis of wart virus infection was made (64.7%, 17) and in patients with mild dyskaryosis (75%, 12)., Conclusions: The methodology evolved localises HPV sequences directly to epithelial cell nuclei, which can be morphologically assessed by haematoxylin counterstaining. Sample contamination with exogenous viral sequences can be distinguished from true infection. In this study, a HPV signal was not found in morphologically normal epithelial cells. The methods described will permit the detection of HPV sequences in routinely collected cervical smears and the evaluation of the natural history and potential clinical relevance of HPV infection without changes in clinical practice.

Aims: To determine which type of human papillomavirus (HPV) is associated with cervical adenocarcinoma and whether the virus was integrated or episomal in two continents., Methods: Biopsy specimens from the UK (n = 16) and South Africa (n = 22) were analysed by non-isotopic in situ hybridisation (NISH) for HPV types 6, 11, 16, 18, 31, 33, and 35 on archival biopsy specimens using digoxigenin labelled probes., Results: A total of 20 adenocarcinomas (53%) from both groups contained HPV DNA. In the UK group, seven and four cases contained HPV 18 (44%) and 16 (25%) respectively. In the South African group, nine cases contained HPV 18 (41%) while HPV DNA was not detectable in the other 13 cases. Hence HPV 18 was present in 80% of HPV positive adenocarcinomas., Conclusions: The HPV 16 or 18 genome was integrated in all viral positive cases. In two cases HPV 18 was also present in an episomal form. These data indicate that HPV integration is common to cervical adenocarcinoma in two continents by the same methodology. The lower prevalence of HPV 18 detection in the South African group may have been due to the presence of other or unsequenced HPV types.

Aims: To determine the relative diagnostic sensitivity of non-isotopic in situ hybridisation (NISH) for the diagnosis of human papillomavirus (HPV) on matched smears and biopsy specimens; to compare the NISH signal type in the two samples; and to correlate the NISH data with the morphological diagnosis., Methods: HPV samples were assayed individually by NISH with digoxigenin labelled probes (HPV6, 11, 16, 18, and 33) on routinely collected paraffin wax embedded cervical biopsy specimens and for high risk HPVs with a cocktail of similarly labelled probes (HPV16, 18, 33) on matched smears. These were taken at the same colposcopic examination from 32 patients investigated for an abnormal cervical Papanicolaou (PAP) stained smear., Results: An HPV signal was present in 18 (56%) biopsy specimens and in 14 (44%) smears. There was higher concordance of sets of data in the presence of cytopathic wart virus changes. The superiority of biopsy over smear in detecting HPV was mainly the result of examining the entire cervical biopsy specimen rather than cells scraped from the cervical surface. The NISH signal type in both biopsy specimen and smear was similar; it has been shown that NISH type 1 signal correlates with episomal viral replication and type 2 and 3 signals with viral integration., Conclusions: These data show that NISH on cervical smears is a worthwhile primary screen for HPV infection. The NISH signal types in cervical smears are similar to those previously described in cervical biopsy specimens.

It was postulated that non-isotopic in situ hybridisation (NISH) signal types 1-3 for human papillomavirus in cervical biopsy specimens represent episomal or integrated virus. The aim of this study was to validate this hypothesis by independent molecular techniques. Fresh cervical intraepithelial neoplasia (CIN) and squamous cell cancer (SCC) tissue were examined for NISH signal pattern by hybridising with digoxigenin labelled HPV 16. DNA was extracted from the same samples and analysed by restriction endonuclease digestion and Southern blotting to determine the physical state of the viral genome. Six CIN biopsy specimens showed a type 1 NISH signal for HPV 16. On Southern analysis these biopsy specimens contained only episomal HPV 16. Three SCC with a type 2 NISH signal contained integrated HPV 16 by Southern analysis. Two specimens, a CIN 3 and an SCC with a type 3 NISH signal for HPV 16, showed the presence of both episomal and integrated HPV 16 with conventional Southern analysis and two dimensional gel electrophoresis. These results show that episomal HPV can be reliably determined by NISH type 1 signal, integrated HPV by type 2, and a combination of both episomal and integrated HPV, by a type 3 signal in archival paraffin wax embedded cervical biopsy specimens. This will add another variable to the epidemiological studies of HPV infection. In particular, it will now allow retrospective studies to be done to define the role of episomal and integrated HPV in the evolution of cervical intraepithelial neoplasia and other cervical disease associated with this virus.

The aim of this study was to optimise conditions for mRNA detection by nonisotopic in situ hybridisation (NISH) using biotinylated and digoxigenin labelled riboprobes. Because lysozyme gene transcripts are present at high concentrations in Paneth and other alimentary cells, archival gut biopsy specimens were chosen as a model system for these experiments. Most of the variables in NISH, from unmasking of mRNA, to its ultimate detection by peroxidase or alkaline phosphatase based detection systems, were examined in detail. The most important findings were that simultaneous heating of tissue targets and riboprobes at 95 degrees C for 15 minutes before hybridisation at 50 degrees C for two hours gave the most intense signal for lysozyme mRNA in Paneth cells, Brunner's glands, and lamina propria macrophages; digoxigenin labelled riboprobes gave a higher signal to noise ratio than their biotinylated counterparts, and probes 600 base pairs long were superior to shorter probes. It is concluded that the mRNA NISH method may be generally useful for detecting gene transcription in archival clinical biopsy specimens.

In a previous study three types of HPV signal were described in CIN. It was suggested that a type 1 signal represented episomal HPV while a type 2 signal represented integrated HPV; and a type 3 signal was indicative of both episomal and integrated HPV. To test this hypothesis 91 squamous cell cancers (SCC) of the cervix from Britain and South Africa were examined for HPV 6, 11, 16, 18, 31, 33, 35. Of the South African group (n = 69) 64% contained HPV types 16 (n = 29) and 18 (n = 15). The SCC in the British group (n = 22) contained HPV 16 and HPV 33 in 12 and three cases, respectively. Of the HPV positive biopsy specimens, 86% showed a type 2 signal in keratinising and non-keratinising tumours and the remainder a type 3 signal. Type 3 signal was present only in keratinising tumours. The presence of punctate signal in 100% of HPV containing SCC, together with localisation of HPV signal to sister chromatids in tumour cell mitotic figures in vivo, provides further evidence for type 2, and the punctate component of type 3 signal representing viral integration.

In South Africa asymptomatic wart virus infection diagnosed by morphological criteria occurs in 16-20% of all ethnic groups; the incidence in black women is 66%. To identify human papillomavirus (HPV) types the prevalence of HPV in cervical intraepithelial neoplasia (CIN) in South African women (n = 72) with age matched British women (n = 73) was compared by non-isotopic in situ hybridisation (NISH) using digoxigenin labelled probes for HPV 6, 11, 16, 18, 31, 33 and 35 on archival biopsy specimens. A higher proportion of British biopsy specimens (68%) contained HPV than those from South Africa (50%) in CIN 2 and 3; this difference was due to HPV 16. Thirty six per cent of the positive biopsy specimens from South African women also contained HPV 33/35 compared with 16% in the United Kingdom. There was no difference in HPV detection with age in either group. These data indicate that HPV types vary geographically, with "minor" HPV types being more common in South Africa. Three qualitatively distinct NISH signals were observed; a diffuse (type 1) signal in superficial cells, mainly koilocytes; a punctate signal (type 2) in basal/"undifferentiated" cells in CIN 3; and combined type 1 and 2 signals in CIN with wart virus infection (type 3). The punctate signal may represent HPV integration.

The sensitivity and specificity of immunocytochemistry were compared with those of non-isotopic in situ hybridisation (NISH) for the direct detection of human papillomaviruses in biopsy specimens. Four monoclonal antibodies raised to the capsid protein of HPV16 were less specific than NISH: all four reacted with lesions containing HPV33, and HPV18. Absolute discrimination of HPV types, therefore, was not possible with the monoclonal antibodies used in this study. The relative sensitivities of these antibodies were also lower than NISH. Sequential immunocytochemistry and NISH on the same section showed that 2.9-13.0 times as many cells were positive by NISH than by immunocytochemistry using the most sensitive monoclonal antibody. These data indicate that NISH has higher diagnostic specificity and sensitivity than immunocytochemistry using monoclonal antibodies to the HPV16 capsid protein.

A monoclonal antibody to digoxin enabled sandwich techniques to be used for the detection of hybridised digoxigenin labelled probes in cultured cells and paraffin wax sections. This system has greater flexibility than alkaline phosphatase conjugated polyclonal antidigoxigenin antibody and permits the use of alternative detector enzymes, such as horseradish peroxidase and fluorescence labels. The APAAP detection system that does not require the use of biotin can also be used in situations where endogenous biotin is a problem. The low level of background staining combined with precise substrate deposition of the amplified peroxidase system gives higher sensitivity and resolution. This permits localisation of closely adjacent chromosomal loci in interphase nuclei. The most sensitive peroxidase based digoxigenin detection system visualises two and a half to 12 copies of human papillomavirus (HPV) per nucleus. This system is also suitable for the analysis of low copy number HPV infection of cervical tissues.

A method was developed for the simultaneous detection of viral and human DNA in contrasting colours in routine formalin fixed, paraffin wax embedded biopsy specimens. This was achieved by non-isotopic in situ hybridisation (NISH) with a biotinylated Y chromosome probe and digoxigenin labelled probe for human papilloma virus type 6 (HPV 6). The tissues studied were peripheral lymphocytes, tonsil, and penile warts. The hybridisation signals produced by biotinylated probes were visualised in red using streptavidin peroxidase and those produced by digoxigenin labelled probes as a blue/black colour using anti-digoxigenin alkaline phosphatase. In lymphocytes and tonsil 95-100% of cells had a detectable Y chromosome; in warts only 60-70% of infected keratinocytes near the skin surface had a demonstrable Y chromosome. This suggests that this chromosome is lost or occluded in cell maturation. In simultaneous double hybridisation with both probes, HPV and Y sequences were demonstrable within the same nucleus in penile warts. This technique permits the simultaneous differential detection of two nuclei acid sequences in interphase nuclei and will have application in analysis of putative dual HPV infections and in determining the intranuclear spatial relations between nucleic acids in interphase nuclei.

This study was undertaken to develop technology for the detection of nucleic acid using two different DNA probe reporter molecules, the ultimate aim being to differentially label two nucleic acids within the same nucleus. Digoxigenin and biotin were used to label DNA probes. The absolute and relative sensitivity of digoxigenin and biotin labelled DNA probes for detecting integrated human papilloma virus 16 (HPV16) was investigated in CaSki cells by non-isotopic in situ hybridisation (NISH). Several methods for the detection of labelled probes were also investigated. The optimal sensitivity of digoxigenin labelled probe was equivalent to that of biotin when alkaline phosphatase was used as the final detector. The median number of discrete viral signals discernible in each cell with the most sensitive detection system was seven to eight with both labelled probes. The average number of HPV16 genomes in each CaSki cell, derived by dot blot hybridisation, was about 270. The calculated absolute sensitivity of NISH for viral detection in this system is complex because of variation of signal size and number. Nevertheless, one signal per nucleus equates to as little as 30 to 40 viral copies, and probably much less. The ability to distinguish up to 15 discrete signals with both digoxigenin and biotin labelled probes in the nuclei of CaSki cells indicates that these methods will be useful in interphase cytogenetics in material routinely fixed in aldehyde.