Detection of high-risk subtypes of human papillomavirus in cervical swabs: routine use of the Digene Hybrid Capture assay and polymerase chain reaction analysis.

Human papillomaviruses (HPVs) are major causative agents in the pathogenesis of cervical cancer, and more than twenty types are associated with its development. With the introduction of liquid-based preparation systems, it is envisaged that large-scale HPV testing will be established in the near future. Preliminary studies demonstrate the accessibility of these samples for DNA testing using both the Digene Hybrid Capture assay (DHCA) and polymerase chain reaction (PCR) techniques. This study aims to assess the validity and sensitivity of the DHCA system to detect high-risk HPV DNA, using two sets of HPV consensus primers (Gp5+/Gp6+ and MY09/MY11) in tandem with routine assessment of cervical smear and biopsy samples. Results indicate that the combination of DHCA and PCR detects more high-grade lesions than does the DHCA alone. DHCA-negative cases were categorised by subsequent PCR amplification into low-grade HPV-negative (12/16) cervical lesions and high-grade HPV-positive (7/9) cervical lesions. Gp5+/Gp6+ primers were less sensitive in detecting HPV-positive samples than was the MY09/MY11 primer set. These results support the use of high-risk HPV testing by DHCA, with subsequent analysis of DHCA-negative samples by PCR using the MY09/MY11 primers.

Human papillomavirus and mixed epithelial tumors of the endometrium.

Strong epidemiological evidence links human papilloma viruses (HPV) with the development of cervical intraepithelial neoplasia (CIN) and invasive cancers of the uterine cervix. The localization of HPV DNA sequences high up in the female genital tract (in benign and malignant lesions) is not that uncommon, but its precise significance is uncertain. In particular, the detection of HPV DNA sequences by polymerase chain reaction (PCR) needs careful interpretation, because the source of the amplicon may emanate from tumor cells, direct contamination from the cervix, or possibly from extratumoral sites in the endometrium. We have previously reported the identification of koilocyte-like changes in the squamous epithelium of some endometrial adenoacanthomas. Adenoacanthomas (adenocarcinoma with squamous metaplasia) are mixed epithelial tumors arising in the endometrium composed of malignant glandular areas admixed with benign metaplastic squamous epithelium. The rarer adenosquamous carcinoma containing both malignant glandular and squamous areas is also described. The origin of benign/malignant squamous epithelial islands in endometrial tumors has been the subject of speculation, with some investigators considering an origin from metaplastic glandular endometrial cells. In this study, we examined 10 normal endometrial samples, 20 adenocarcinomas, 41 adenocarcinomas with squamous metaplasia, and two adenosquamous carcinomas, (including control cervical material where possible) for the presence of HPV DNA sequences using nonisotopic in situ hybridization (NISH), type-specific HPV PCR, general primer PCR (to detect sequenced and unsequenced HPVs), and PCR in situ hybridization (PCR-ISH). We did not identify HPV DNA sequences in normal endometrial tissue. In adenocarcinomas (endometrioid type), HPV was only identified in 2 of 20 cases by PCR, both of which were HPV 11 positive. We were unsuccessful in identifying HPV in endometrial carcinomas by NISH or by PCR-ISH, raising the possibility of contamination from the cervix in the two positive cases. In adenoacanthomas, a low-risk HPV type (HPV 6) was found in 19 of 41 cases. NISH signals were intranuclear in location in squamous regions of adenoacanthomas. Additional positive nuclei were uncovered using PCR-ISH, which increases the sensitivity of standard NISH detection. HPV DNA sequences were located in some malignant endometrial glandular epithelial cells, but this accounted for a minority of samples. HPV DNA sequences were not detected in extraepithelial sites. Mixed infection by two different HPV types was identified in two cases. Most cases showed similar HPV types in cervical and endometrial lesions, although discordant cases were uncovered. In adenosquamous carcinomas, one case showed mixed infection with HPV 6 and 33 by PCR. The apparent segregation of low-risk HPV type (HPV 6) with benign squamous metaplastic epithelium in adenocarcinoma with squamous metaplasia, and high-risk type (HPV 33) with malignant squamous epithelium in adenosquamous carcinoma, raises important questions in relation to the role of HPVs in mixed epithelial tumors of the endometrium and their interplay in the pathogenesis of squamous metaplasia at extracervical sites.

Perianal hidradenoma papilliferum occurring in a male: a case report.

Hidradenoma papilliferum is a rare apocrine gland tumour, described only once previously in a male. We present the second such case.

Alterations in exon 1 of c-myc and expression of p62c-myc in cervical squamous cell carcinoma.

AIMS: To examine human papillomavirus (HPV) positive and negative squamous cell carcinomas of the cervix for structural alterations in exon 1 c-myc; and to investigate the expression pattern of p62, the protein product of c-myc. MATERIAL: Archival paraffin wax embedded tissues of cervical squamous cell carcinomas, stage I and II, retrieved from the files of the department of pathology, University College Cork, Ireland: 40 cases were examined for alterations in exon 1 of c-myc; 57 cases were used for immunocytochemical p62 analysis. METHODS: c-myc exon 1 PCR on HPV positive and negative stage I and II cervical squamous cell carcinomas was performed using primers designed to fragile sites in exon 1 of the c-myc oncogene, which are frequently involved in translocation phenomena and deletions in other neoplasms. This region is bordered by two promoter sequences P1 and P2. In addition, the expression of p62 was evaluated using the monoclonal antibody Mycl-9E10. RESULTS: Alterations in exon 1 of c-myc were shown in 7.5% of squamous cell carcinomas of the cervix. Changes in exon 1 and 2 of c-myc were also found in COLO 320 cells and Raji cells. These alterations were due to small deletions within exon 1 of c-myc, but point polymorphisms occurring within the priming sites (in one case) may also have occurred. The alterations uncovered appeared "clonal," as replicate samples showed the same amplicon band pattern. Expression of c-myc was variable, with cytoplasmic staining patterns predominating. All cases which showed exon 1 alterations were HPV positive and had strong nuclear positivity on p62 immunocytochemistry. CONCLUSIONS: Alterations in exon 1 of c-myc occur in a minority of cervical cancers and there was increased expression of p62 in a cohort of HPV positive and negative cervical squamous cell carcinomas. Exon 1 alterations may provide an alternative route to c-myc activation in early squamous cell carcinoma.

Genotypic mapping of HPV and assessment of EBV prevalence in endocervical lesions.

AIMS: To examine the prevalence of human papillomavirus (HPV) and Epstein-Barr virus (EBV) in low grade glandular intraepithelial lesions of the cervix, adenocarcinoma with high grade glandular intraepithelial lesions combined, and adenocarcinomas; and to perform a genotyping mapping analysis of endocervical carcinomas to determine the extent of HPV infections in such lesions. MATERIAL: Archival paraffin wax embeded material from the files of the departments of pathology, National Maternity Hospital, Dublin, and University College Cork, Ireland. METHODS: HPV prevalence was examined using type specific HPV PCR, general primer HPV PCR (pan HPV screen), nonisotopic in situ hybridisation (NISH), and PCR in situ hybridisation (PCR-ISH). In situ hybridisation was performed using fluorescein labelled oligonucleotide cocktail for eber transcripts of EBV. Genotypic analysis was performed, in all cases where possible, using a grid system. RESULTS: HPV 16 and 18 were predominantly identified in low grade glandular intraepithelial lesions, high grade glandular intraepithelial lesions, and adenocarcinomas, with HPV prevalence increasing with grade of dysplasia. EBV was only identified in subepithelial lymphocytes in a minority of cases. No link could be shown between HPV and EBV in endocervical lesions. HPV infection was not clonal in endocervical cancer and coexistent adjacent cervical intraepithelial neoplasia, where present, tended to show a similar HPV type. CONCLUSIONS: The restriction of HPV types 16 and 18 to endocervical lesions suggests that their effect is restricted and specific to endocervical mucosa, but the mechanism of interaction is currently unknown.

Epstein-Barr virus in normal, pre-malignant, and malignant lesions of the uterine cervix.

AIM--To detect the presence or absence of Epstein-Barr virus (EBV) in cervical lesions ranging from normality to invasive malignancy. METHODS--Eighteen randomly selected cases of invasive squamous cell carcinomas of the uterine cervix were examined as well as 25 cases each of normal cervices and those showing cervical intra-epithelial neoplasia (CIN) I, II, and III. DNA-DNA in situ hybridisation, using a biotinylated probe to the Bam H1 "W" fragment of EBV, was carried out in addition to the polymerase chain reaction using specific primer sequences that flank a 153 base pair segment of the Bam H1 "W" region of the EBV genome and which do not cross-amplify other DNA herpes viruses. Positive control material included paraffin wax embedded P3 HR1 lymphoblastoid cells (containing high copy numbers of EBV) and two nasopharyngeal carcinomas positive for EBV. RESULTS--Neither normal nor CIN I tissue was positive. Eight per cent of CIN II tissue was positive; 8% of CIN III, and 43% of carcinomas were positive for EBV. CONCLUSION--The study shows that the virus is present in some cases of cervical carcinoma and to a lesser degree in some premalignant lesions of the cervix, but the exact association between it and cervical oncogenesis, be it causative or incidental, remains to be determined.

Seminal vesicle amyloidosis: morphological, histochemical and immunohistochemical observations.

Subepithelial deposits of amyloid were detected within the seminal vesicles of 13 males from a total of 143 unselected autopsies (9%). The incidence increased with increasing age. The amyloid was classified using histochemistry, immunohistochemistry and clinical features. Eight cases were categorized as senile vesicle amyloid, two as systemic AA amyloid with secondary involvement of the seminal vesicle, and three as mixed amyloidosis. The morphological appearances of the different categories of seminal vesicle amyloidosis are similar but a different distribution is common. The staining characteristics of senile vesicle amyloid suggest that this is a different amyloid protein, perhaps locally derived within the seminal vesicle.

Placental chorioangiosis associated with foetal death--a case report.

A case of foetal death with retroplacental haemorrhage at 24 weeks gestation is reported, where the placenta histologically showed diffuse severe chorioangiosis. The latter is an infrequently reported placental vascular anomaly, which recent studies have indicated to be an important sign of neonatal morbidity and mortality.

Adenoacanthoma of the endometrium: morphological changes induced by human papillomavirus.

The observation of koilocyte-like features in the squamous epithelium of some endometrial adenoacanthomas prompted an investigation into a possible viral aetiology. These changes closely resemble those that occur in the ectocervical mucosa which are accepted as morphological evidence of human papillomavirus (HPV) infection. Sections of 87 hysterectomy specimens removed for endometrial carcinoma over 12 years, together with preoperative curettings, were reviewed for the presence of acanthomatous change and for appearances suggestive of HPV infection. The ages of the women ranged from 36 to 84 years, average age 62.6. Light microscopical examination showed koilocytosis, papillary formations, and intranuclear eosinophilic inclusions of both squamous and glandular epithelium in some tumours. Immunocytochemistry and DNA in situ hybridisation indicated the presence of HPV antigen in squamous and glandular cells, and perinuclear virus particles characteristic of HPV were seen on electron microscopical examination in those cells with nuclear inclusions. HPV probably infects endometrial adenocarcinomas directly from the cervix but it is unlikely that it has an aetiological role. It is possible, however, that in addition to being a "passenger," the virus may stimulate squamous metaplasia in some adenocarcinomas of the endometrium and may also exert some influence on their behaviour.

Primary hyperoxaluria--a case report.

A case of primary hyperoxaluria which presented with progressive renal failure in a 34 year old woman is reported. The patient died six years following the initial diagnosis and post mortem examination revealed widespread deposition of oxalate crystals in her tissues.

Percutaneous fine needle aspiration of pulmonary mass lesions--a critical examination of the accuracy of cell typing of malignant tumours.

All the biopsies of pulmonary mass lesions obtained by percutaneous needle aspiration over a seven year period were reviewed. Emphasis is laid upon the differences in cell typing of malignant tumours when two types of preparation of the same tissue, cytological smears and tissue sections are examined. Some problems in cell typing are discussed and suggestions for improved proficiency are made.