Survey of histologic specimens of human cancer for human papillomavirus types 6/11/16/18 by filter in situ hybridization.

Histologic specimens (317) of genital and nongenital cancers and normal tissue were analyzed for the presence of the DNA of human papillomavirus (HPV) 6, 11, 16, and 18 by filter in situ hybridization performed on paraffin-embedded, formalin-fixed tissue (HISTOFISH). HPV DNA was found in 73 of 172 (42%) anogenital lesions and 17 of 116 (15%) nonanogenital carcinomas. No HPV DNA was found in normal mouse skin (five samples), human autopsy liver (two samples), or kidney (eight samples), or in carcinomas of the breast (three samples), bladder (five samples), or colon (nine samples). Of the nongenital tumors, HPV DNA was found in the carcinomas of the lung (2 of 5), anus (7 of 18), esophagus (9 of 39), buccal cavity (1 of 5), and larynx (5 of 50). HPV DNA was also detected in 2 of 11 histologically normal specimens of the cervix and 1 of 3 human skin lesions. The detection of HPV DNA in carcinomas of the lung, larynx, and esophagus as well as in the anogenital region confirms recent suggestions that HPV types 6, 11, 16, and 18 have a wider association with different types of cancer than previously believed. The study also shows that HISTOFISH is a useful method for detecting HPV-DNA in histologic specimens.

Detection of human papillomavirus in reprocessed routine Papanicolaou smears by DNA hybridization.

DNA dot blot hybridization was used for the detection of human papillomavirus (HPV) types 6, 11, 16, and 18 in reprocessed routinely collected Papanicolaou smears. DNA was extracted from the smears with alkaline lysis and applied onto a nitrocellulose filter. The specificity and sensitivity of the dot blot hybridization on reprocessed Papanicolaou smears were confirmed by Southern blot analysis of selected samples, using CaSki, SiHa cell lines as positive controls and HF 32 as negative controls. From 42 normal smears and 44 abnormal smears with koilocytosis present, 9 (21%) and 43 (98%), respectively, were positive for HPV 6/11/16 or 18. These results show that reprocessed Papanicolaou smears in combination with DNA hybridization have potential application in retrospective studies on the prevalence and distribution of HPV infection.

Detection of human papillomavirus type 16 DNA in cervical swabs and formalin-fixed, paraffin-embedded squamous cell carcinomas of non-genital tissues using a synthetic oligodeoxynucleotide probe.

The potential of using a chemically synthesized oligodeoxynucleotide as a diagnostic probe to detect human papillomavirus type 16 (HPV-16) in genital infections was evaluated by comparing it with a cloned full-length HPV-16 probe in dot-blot DNA hybridizations. An oligonucleotide sequence, 20 bases in length from the E6 region of HPV-16 (E6 oligo) and different from the DNA sequences of HPV types 6, 11 and 18 by at least 2 base pairs, was chosen for chemical synthesis. The oligoprobe, which was 5'-end labelled with [32P]dATP, was found to be specific, but approximately ten times less sensitive than the full-length radiolabelled probe of HPV-16, in dot-blot hybridizations with the DNA of HPV-6, -11, -16 and -18, HPV positive and negative cell-lines. From 36 cervical or vulval scrapes two samples were found positive with both cloned HPV-16 and oligoprobe hybridization. Of 21 samples of formalin-fixed, paraffin-embedded squamous cell carcinomas originating from anus, oesophagus, penis, colon, breast and skin only 4 anal squamous cell carcinomas were positives when hybridized with cloned HPV-16 DNA or with the oligoprobe. This study confirms that HPV-16, which is frequently associated with squamous cell carcinoma of the cervix is also strongly associated with squamous cell carcinoma of the anus.

Human papillomavirus coinfections of the vulva and uterine cervix.

DNA filter in situ hybridisation (FISH) was used to determine the presence of human papillomavirus (HPV) genotypes 6/11, 16/18, and 31/33 in cell scrapes of the cervix and vulva of 128 women who had precancerous lesions and/or HPV infection of the cervix diagnosed by cytology, colposcopy, and histology. HPV-DNA was found in 87 (68%) vulval and 95 (74%) cervical cell scrapes, and in both the vulval and cervical scrapes of 73 (57%) women, but not in either the vulva or the cervix of 19 women (15%). Of the HPV-DNA-positive smears, the prevalence of the HPV types was 61% HPV 16/18, 14% HPV 6/11, 3% HPV 31/33, and 22% HPV 6/11 and 16/18. By contrast, HPV-DNA was not detected in the cervical smears of a control group of 35 women who were assessed to be free of cervical abnormalities by colposcopy and cytology. The epithelial response of the vulva and the cervix to application of 5% acetic acid was assessed by colposcopy and the results correlated with the presence of HPV genotypes. A possible or definite disorder of the cervix and vulva was detected by colposcopy in 95 (74%) and 96 (75%) of the 128 cases, respectively. The colposcopic assessment of the vulva was inconclusive in ten cases (8%), and only eight women (6%) were found to be free of both a vulval and cervical disorder. This study shows subclinical papillomavirus infections of the vulva frequently coexist with HPV infections and precancerous lesions of the cervix.(ABSTRACT TRUNCATED AT 250 WORDS)

Detection of human papillomavirus DNA in cell scrapes and formalin-fixed, paraffin-embedded tissue of the uterine cervix by filter in situ hybridisation.

Filter in situ hybridisation (FISH) was used to detect the presence of DNA of human papillomavirus (HPV) types 6/11 or 16/18 in cell scrapes (CYTOFISH) and formalin-fixed, paraffin-embedded biopsies (HISTOFISH) taken from the uterine cervices of 19 women. Paraffin tissue sections collected for HISTOFISH were either digested with pepsin or lysed with alkali/Triton X-100. The digest or lysate of the tissue sections and cell scrapes were applied to nylon or nitrocellulose membranes for nucleic acid hybridisation using 32P-labeled HPV-DNA probes. CYTOFISH and HISTOFISH were compared directly by taking samples for each method from the cervices of the same women. Of 19 women examined by colposcopy, cytology, and histology, eight were assessed as normal and 11 had evidence of a cervical disorder and/or the presence of HPV infection. Whereas no HPV-DNA was detected in the normal cases, the presence of HPV-DNAs was detected by both CYTOFISH and HISTOFISH in 11 cases with histological evidence of HPV infection and/or dysplasia. In these HPV positive cases, eight contained HPV 16/18, two HPV 6/11, and one a mixed infection of HPV 6/11/16/18. The high correlation between the results of CYTOFISH and HISTOFISH shows that formalin-fixed, paraffin-embedded cervical biopsies are suitable specimens for the detection and typing of HPV-DNA by FISH. Both CYTOFISH and HISTOFISH should facilitate studies on the prevalence and distribution of HPVs and their association with neoplasia.

Detection of DNA of human papillomavirus types 6/11 and 16/18 in cell scrapings of the uterine cervix by filter in situ hybridisation. Correlation with cytology, colposcopy and histology.

The application of filter in situ hybridisation (FISH) to detect the presence of the DNA of human papillomavirus genotypes 6/11 and/or 16/18 in cell scrapings of the uterine cervix of 248 women in Western Australia is described. The results obtained by FISH are related to cervical dysplasia as assessed by cytology, colposcopy and histology. The detection of HPV infection was more sensitive and specific by FISH than by either histological/cytological evidence of an HPV cytopathic effect (koilocytosis) or immunohistochemical staining for HPV capsid antigen using antiserum against genus specific antigen of the bovine papillomavirus. Viral DNA was detected by FISH in 65% of women with atypical cytology and of the HPV positive cases, 68% were HPV 16/18, 22% HPV 6/11 and 10% of mixed types. HPV-DNA was detected in the cervical smears of 16 women who also had HPV capsid antigen (HPV-Ag) in their cervical biopsies; HPV 6/11 was found in 4 cases and HPV 16/18 in 12 cases. The relative frequency of the HPV-Ag positive cases decreased markedly from 44% to 4% with an increase in the severity of cervical dysplasia. By comparison, the percentage of HPV-DNA positive cases remained relatively constant between 70% and 80% for all 3 categories of dysplasia. Of these, the percentage of HPV 6/11 positives decreased slightly with an increase in the severity of dysplasia as assessed by either cytology or histology, whereas the percentage of HPV 16/18 positive cases was relatively constant. HPV-DNA was found in cervical smears of 11 of 48 (23%) women who were diagnosed colposcopically to have atypical transformation zone of the cervix. In 138 women with an atypical transformation zone, 96 (70%) were found to have HPV-DNA with HPV 16/18 contributing to 83% of the HPV positive cases. All four genotypes of HPV were found to be associated with the colposcopic morphology of mosaicism (26 cases) but only HPV 16/18 was found in 12 HPV-DNA positive cases associated colposcopically with punctation and an atypical transformation zone. The detection and typing of HPV-DNA in cell scrapings by FISH is a relatively fast and non-invasive procedure which complements cytology, colposcopy and histology and should be useful in further studies of the natural history of different HPV infections and their role in cervical cancer.

DNA sequences of human papillomavirus types 11, 16 or 18 in invasive cervical carcinoma of Western Australian women.

The presence of human papillomavirus (HPV) types 11, 16 and 18 in 77 biopsies of cervical intraepithelial neoplasia (dysplasia) and carcinoma of the uterine cervix of a sample of women from Western Australia was examined using "Southern" blot hybridisation. HPV-DNA was found in 17 of the 23 dysplasias and 43 of the 54 invasive carcinomas examined but not in the 5 biopsies obtained from areas assessed as normal by colposcopy and histology. Five of 11 biopsies of mild to moderate dysplasias contained HPV type 11 (HPV-11), 2 HPV-16 and 1 HPV-18. Of 12 severe dysplasias/carcinoma in situ, 2 contained HPV-11, 4 HPV-16 and 2 HPV-18. One biopsy contained both HPV-11 and HPV-16. Of 45 squamous cell carcinomas examined for HPV-DNA, 24 contained HPV-16, 5 HPV-11 and 1 HPV-18. Both HPV-11 and HPV-16 were found in 6 of the squamous cell carcinomas and 2 contained both HPV-16 and HPV-18. Of 6 adenosquamous carcinomas examined, 3 contained HPV-DNA, 2 with HPV-16 and 1 with HPV-11. HPV types 16 or 18 were also found in 2 of 3 adenocarcinomas. This study shows a strong association between the papillomavirus and uterine cervical cancer in a sample of women from Western Australia. HPV-16 was more frequently associated with severe dysplasia and cancer than with mild or moderate dysplasia supporting the view that this HPV genotype may have a greater oncogenic potential than HPV-11.

Comparison of peroxidase-antiperoxidase and avidin-biotin complex methods for the detection of papillomavirus in histological sections of the cervix uteri.

A study was undertaken to determine the relative sensitivities of the peroxidase-antiperoxidase (PAP) and avidin-biotin complex (ABC) methods for the detection of human papillomavirus (HPV) antigens in acetic acid-ethanol fixed paraffin-embedded cervical tissue. Tissue sections prepared from 14 women suspected to have HPV infections with either atypia or dysplasia were stained immunohistochemically using an antiserum against genus-specific (common) antigen of bovine papillomavirus. Detection of HPV antigen was approximately twice as frequent by the ABC method as by the PAP method. Of the 14 cases studied, 43% were found to be HPV positive by the PAP method whereas 79% were HPV positive by the ABC method. In addition, the number of cells found to be HPV positive by the ABC method was approximately double the number by the PAP method.