In situ hybridization analysis of human papillomavirus in anal squamous cell carcinoma.

To investigate the association between human papillomavirus (HPV) infection and anal carcinoma, we applied a sensitive in situ hybridization technique to detect HPV messenger RNA (HPV m-RNA) in formalin-fixed, paraffin-embedded sections from 18 patients. Using tritium-labeled probes, HPV m-RNA was detected in 12/18 (67%) patients. HPV 6 was detected in four patients, coexisting with HPV 18 in two cases, and HPV 16 was found in eight patients. In six patients, hybridization failed to demonstrate the presence of HPV. With respect to histology, HPV 6 was detected in 1/4 cases of well differentiated invasive squamous cell carcinoma. Ten of thirteen moderately or poorly differentiated invasive squamous cell carcinomas demonstrated HPV m-RNA (HPV 16, eight cases; HPV 6, one case; HPV 6 and 18, one case). HPV 31 was not detected in any specimens. These results suggest that HPV infection may play an important role in the pathogenesis of anal carcinoma.

Male partners of women with genital human papillomavirus infection. An assessment of colposcopic abnormalities by histological examination and human papillomavirus hybridization.

Men whose female sexual partners showed histological evidence of human papillomavirus infection were examined. Human papillomavirus DNA was identified in 29 of 35 biopsy samples of colposcopically-identified penile lesions. Human papillomavirus strains that were related to human papillomavirus genotypes 6/11 were observed most commonly (seven of eight patients) in the partners of patients with warty atypia or condylomata, while human papillomavirus strains that were related to human papillomavirus genotypes 16/18 were most-commonly (eight of 15 patients) observed in tissue from the partners of patients with cervical intraepithelial neoplasia. Measurement of human papillomavirus DNA in lesions by the filter in-situ hybridization technique more-frequently indicated human papillomavirus infection (29 of 35 lesions) than did conventional histopathological assessment (21 of 35 lesions) in this "high-risk" group. We conclude that colposcopically-identifiable lesions in male sexual partners are likely to contain human papillomavirus DNA, even if is no definite histological evidence of human papillomavirus infection is present, and that such lesions frequently contain strains of human papillomavirus that have been associated with the development of anogenital carcinoma.

Nonhematopoietic tumor cells express functional GM-CSF receptors.

Human granulocyte-macrophage colony-stimulating factor (GM-CSF) stimulates the colony growth of myeloid progenitors in semisolid media, and enhances the function of mature effector cells, including neutrophils, monocytes, and eosinophils. Small cell carcinoma lines (SCCL) have properties of amine precursor uptake and decarboxylation (APUD) cells and express high levels of the enzyme, L-aromatic amino acid decarboxylase. We looked for possible expression of GM-CSF receptors on nonhematopoietic cells and found specific high-affinity binding of human GM-CSF to SCCL and to the SV40-transformed African green monkey kidney cell line, COS. The small cell carcinoma lines responded to GM-CSF with enhanced proliferation, and both small cells and COS cells were found to express authentic 84,000 dalton GM-CSF receptor protein. These findings indicate that nonhematopoietic cells can bind and respond to GM-CSF, suggesting additional biological activities as well as the possibility of tumor responses when GM-CSF is used therapeutically in humans. Since preliminary clinical trials using CSFs as adjunctive treatment in patients with solid tumors are underway, it will be important to consider the possible responsiveness of nonhematopoietic tumor cells to CSFs.

In situ hybridization and immunohistochemical study of human papillomavirus infection in adult laryngeal papillomas.

Routinely processed paraffin sections from 20 patients with adult laryngeal papillomas were examined for the presence of human papillomavirus type 11 (HPV-11) DNA and its specific mRNA by in situ hybridization methods using 35S-labeled RNA probes. Immunohistochemical techniques were also used to identify papillomavirus genus-specific common antigen (pgs-antigen). HPV-11 DNA signals and/or papillomavirus genus-specific common antigen were detected in all eight samples of multiple laryngeal papilloma. On the other hand, in 12 samples of single laryngeal papilloma, neither papillomavirus genus-specific common antigen nor HPV-11 DNA were detected. Four patients were positive for both HPV-11 DNA and pgs-antigen. In three of these four patients, HPV-11 mRNA signals were also detected. These results provided direct evidence of the association of HPV and adult multiple laryngeal papilloma.

Human papilloma virus infection and skin cancer in renal allograft recipients.

202 renal allograft recipients in south-east Scotland, who had received transplants between 1965 and 1986, were monitored over 3 years (1984-87) for the presence of warts, keratoses, and skin cancers. 77% of 69 patients with graft survival of more than 5 years had viral warts, 38% had keratoses, and 12% had skin cancers, whereas of the 133 with graft survival of less than 5 years 20% had warts, 17% had keratoses, and 1.5% had skin cancers. The ratio of squamous cell carcinoma to basal cell carcinoma was 15:1. Most viral warts showed significant epidermal dysplasia, and keratoses and squamous cell carcinomas had signs of human papilloma virus infection. 15 (60%) of 25 squamous cell carcinomas contained HPV5/8 DNA and 1 contained HPV4 DNA--HPV5/8 DNA was detected in skin lesions of recipients with cancers significantly more often than in those matched for duration and type of immunosuppression with nonmalignant skin lesions. The findings suggest a role for HPV5/8 in the aetiology of squamous cell carcinoma in renal allograft recipients.

Detection of human papillomavirus deoxyribonucleic acid in exfoliated cervicovaginal cells as a predictor of cervical neoplasia in a high-risk population.

Specific types of human papillomavirus are currently implicated as etiologic agents of precancerous and cancerous lesions of the cervix. We have previously described the use of cervicovaginal lavage and molecular hybridization to detect human papillomavirus infections of the cervix. We report here the predictive value of this method of human papillomavirus detection to identify women with biopsy proved dysplastic and cancerous lesions of the cervix. One hundred ninety-one women from a city hospital colposcopy clinic underwent concurrent Papanicolaou smear, cervicovaginal lavage, and coloposcopically directed cervical biopsy. Human papillomavirus deoxyribonucleic acid was detected in 114 (59.7%) of these women. Of the positive results, human papillomavirus type 16 accounted for 23.7%, human papillomavirus type 18 for 10.5%, human papillomavirus type six or 11 for 6.2%, related human papillomavirus types for 52.6%, and 7.0% contained more than one type. The distribution of human papillomavirus types was similar in both women younger than 40 years of age and in older women. Eighty-nine of 128 (69.5%) women less than 40 years old with cervical lesions had positive findings of human papillomavirus, and 18 of 29 (62.1%) older women with cervical lesions had positive findings of human papillomavirus. Detection of human papillomavirus types 16 and 18 identified only 35 of 157 (22.3%) women with cervical lesions. The sensitivity of detecting all types of human papillomavirus as a predictor of a biopsy proved lesion (68.0%) was comparable with the sensitivity of cytologic examination alone (74.0%). However, human papillomavirus detection combined with the Papanicolaou smear provided an increased overall sensitivity of 89.3% (p less than 0.01). In fact, women either positive for human papillomavirus or having abnormal cytologic findings were 11.8 times more likely to have a biopsy proved cervical lesion than human papillomavirus-negative women with negative cytologic results (95% confidence interval for odds ratio: 5.3 to 26.6). We conclude that the sensitivity of cytologic examination plus human papillomavirus detection is superior to the use of either cytologic studies or human papillomavirus detection alone in identifying patients with cervical lesions.

Screening of premalignant cervical lesions for HPV 16 DNA by sandwich and in situ hybridization techniques.

A series of 97 cervical smears and 69 directed punch biopsies derived from 84 consecutive women prospectively followed-up for cervical HPV (human papillomavirus) infections were studied using the sandwich hybridization and in situ hybridization techniques with HPV 16 DNA probes. The aim was to test the sensitivity and applicability of these two techniques in routine diagnosis of cervical HPV infections from smears. As a measure of specimen adequacy, the number of cells recovered in the cervical scrape was determined along with HPV 16 DNA in the sandwich hybridization test using human pro-alpha 2(I)-collagen gene probe. CIN (cervical intraepithelial neoplasia) was suggested in 56% of the patients by the Pap smear, and disclosed in 65% of the biopsies. HPV 16 DNA was present in 57% of cervical scrapes consistent with CIN, i.e., were of Pap smear classes III or IV. Forty percent of the scrapes not suggestive of CIN, i.e., Pap smear classes I or II, also contained HPV 16 DNA. The detection rate for HPV 16 DNA of the sandwich hybridization method was 89% of that of the in situ method in adequate scrapes, but only 43% in cell-poor specimens. The number of HPV 16 DNA-positive scrapes as compared with the total number of diagnoses obtained by studying also the biopsies was 31/36 (69 patients). The results indicate that the cervical scrape as a noninvasive specimen is applicable for screening of cervical HPV infections, and it can be studied with acceptable sensitivity by the rapid sandwich hybridization technique. However, if a punch biopsy is indicated it should be studied using the in situ hybridization technique that allows more sensitive detection of HPV DNA than any other hybridization method and enables the analysis of several HPV types in the same sample instead of only one HPV type in the scrapes.

Studies on the polyoma tumor-specific transplantation antigen (TSTA): selection and characterization of TSTA-negative segregants from somatic hybrids.

Two polyoma-TSTA-negative variants were selected independently from a polyoma fibrosarcoma/Moloney lymphoma somatic hybrid, by repeated passages in polyoma-virus-preimmunized mice. One of the variants had lost all its polyoma DNA, while the other only retained a deleted piece of its integrated polyoma DNA. In contrast to the parental hybrid clone, none of the variants produced detectable amounts of T-antigens. This finding indicates that a detectable expression of the products of the polyoma virus early genome, the T-antigens, is important either directly or indirectly for the expression of TSTA.

HPV DNA detection in tumours of the head and neck: a comparative light microscopy and DNA hybridization study.

A series of 22 squamous cell carcinomas (4 cases grade 1; 11 cases grade 2; 7 cases grade 3) of the oral cavity (13 cases), (naso-)pharynx (5 cases) and larynx (4 cases) were studied by conventional light microscopy and filter (dot blot) hybridization for the detection of human papillomavirus (HPV) DNA. In 4 carcinomas, tumour-free tissue from the resection margins of the surgical sample was examined in addition to the tumour specimen. The same kind of investigation was carried out on 4 oral leukoplakias without dysplasia. All cases were thoroughly examined for HPV-associated cytopathic effects (koilocytosis). In all cases, material was obtained for DNA extraction followed by dot blot hybridization. DNA hybridization was carried out under stringent conditions with mixed probes of HPV 6/11 as well as HPV 16/18. Koilocytosis was observed in 10/22 carcinomas (45%, 4/4 G1 tumours, 6/11 G2 tumours, none out of 7 G3 tumours) and 3/4 leukoplakias. Koilocytosis always occurred at the tumour surface or the surface epithelium immediately adjacent to the tumour. HPV DNA was found in 8/22 carcinomas (36%, 2/4 G1 tumours, 5/11 G2 tumours, 1/7 G3 tumours). We observed HPV 16/18 infections in 3 cases and HPV 6/11 infection in 1 case. The other 4 cases were positive under relaxed conditions and, thus, could not be grouped into one of the examined types of HPV infections. In 4 carcinoma cases, tumour tissues and resection margins were examined. 3/4 cases showed concordant findings, i.e. in 2 cases tumour tissue and tumour-free mucosa (1-2 cm distant to the tumour) were positive for HPV, 1 case was negative in both samples. In 6/8 cases positive for HPV, HPV DNA detection corresponded to the observation of intensive koilocytosis. The leukoplakias were seen to harbour HPV DNA in 3 cases (1 case: HPV 6/11; 1 case: HPV 16/18; 1 case: positive under relaxed conditions). We did not observe HPV DNA in normal mucosal tissues. Our findings provide further evidence for the presence of HPV infections in tumours of the upper respiratory and digestive tract. Prospective studies now have to clarify the biological importance of HPV infections in this group of human cancer.

Correlation of cellular atypia and human papillomavirus deoxyribonucleic acid sequences in exfoliated cells of the uterine cervix.

Restriction enzyme digestion and Southern blot hybridization were used to analyze deoxyribonucleic acid (DNA) extracted from exfoliated cervical cells for the presence of human papillomavirus sequences and these results were correlated with cytologic findings on Papanicolaou smears. Specimens (N = 204) were obtained from a nonselected population of women undergoing routine cytologic screening and human papillomavirus DNA sequences were detected in 33 (16%) women. Thirteen smears contained atypical squamous cells, ranging from very mild dysplasia to moderate dysplasia; all showed associated morphologic evidence of human papillomavirus infection characterized by koilocytosis, nuclear enlargement, wrinkling, and hyperchromasia, and human papillomavirus DNA was demonstrable in 12 (92%) smears. Of the remaining 191 samples with normal cytology, 21 (11%) also contained human papillomavirus DNA sequences. Reevaluation of the smears from these women resulted in a revision of the cytologic diagnosis to very mild dysplasia in four cases. These data suggest that human papillomavirus infection occurs more frequently than predicted by cytologic screening.

Human papillomavirus deoxyribonucleic acid in cervical carcinoma from primary and metastatic sites.

Tissue from 13 cervical cancers and pelvic or para-aortic lymph nodes from the same patient were evaluated by deoxyribonucleic acid hybridization with a human papillomavirus type 16 deoxyribonucleic acid probe for the presence of human papillomavirus-related deoxyribonucleic acid sequences. Twelve of the primary malignancies were squamous cancers and one was an adenocarcinoma. Eight of the primary tumors contained human papillomavirus type 16 deoxyribonucleic acid sequences, and five contained viral sequences closely related to human papillomavirus type 16. Histopathologic diagnosis confirmed malignant cells in six of 13 lymph nodes; three of these specimens contained human papillomavirus type 16 sequences while three had human papillomavirus type 16-related sequences. One lymph node that failed to show malignant cells also contained human papillomavirus type 16 deoxyribonucleic acid. The remaining lymph nodes did not contain malignant cells by either histologic examination or deoxyribonucleic acid hybridization. The human papillomavirus deoxyribonucleic acid sequences in the lymph nodes were similar to those in the matched primary cancer in all cases. These data provide further evidence implicating human papillomavirus in the etiology of cervical cancer.

Bowenoid papulosis. Presence of human papillomavirus (HPV) structural antigens and of HPV 16-related DNA sequences.

This study reviews 39 cases of anogenital bowenoid papulosis lesions in 22 individuals of both sexes that were analyzed clinically, histologically, immunocytochemically, and virologically. Macroscopically, three different types of lesions were demonstrated: erythematous macules; papules (lichenoid and/or pigmented papules); and leukoplakialike lesions. Microscopically, bowenoid papulosis fulfills the criteria of a squamous cell carcinoma in situ. Much like oral precancers, three distinct growth patterns (flat, endophytic, and exophytic) could be differentiated, which did not correlate with the clinical aspect of the lesions. In only two (5.12%) of the 39 cases of bowenoid papulosis could structural antigens of papillomaviruses be detected immunocytochemically (peroxidase-antiperoxidase technique). The DNA from 12 lesions that were analyzed for the presence of papillomavirus-specific sequences hybridized stringently in all cases with the human papillomavirus 16 specific DNA probe labeled with phosphorus 32.

Type beta transforming growth factor from feline sarcoma virus-transformed rat cells. Isolation and biological properties.

We have isolated a strongly mitogenic, type beta transforming growth factor (beta TGF) released by Snyder-Theilen feline sarcoma virus-transformed rat embryo (FeSV-Fre) cells that induces phenotypic transformation of normal NRK cells when they are concomitantly stimulated by analogues of epidermal growth factor (EGF). Molecule filtration chromatography separates beta TGF from an EGF-like TGF (eTGF) which is also present in acid extracts from medium conditioned by FeSV-Fre cells (J. Massagué, (1983) J. Biol. Chem. 258, 13606-13613). Final purification of beta TGF is achieved by reverse phase high pressure liquid chromatography (HPLC) on octadecyl support, molecular filtration HPLC, and nonreducing dodecyl sulfate-polyacrylamide gel electrophoresis steps, yielding a 300,000-fold purified polypeptide with a final recovery of 21%. The purified rat beta TGF consists of two Mr = 11,000-12,000 polypeptide chains disulfide-linked as a Mr = 23,000 dimer. Induction of anchorage-independent proliferation of NRK cells by rat beta TGF depends on the simultaneous presence of eTGF or EGF. In the presence of a saturating (300 pM) concentration of either rat eTGF or mouse EGF, half-maximal anchorage-independent proliferation of NRK cells is obtained with 4-6 pM rat beta TGF. In the presence of a saturating (20 pM) concentration of rat beta TGF, half-maximal anchorage-independent proliferation of NRK cells is obtained with either rat eTGF or mouse EGF at a 50-70 pM concentration. Rat beta TGF is also able to induce DNA synthesis and cell proliferation on growth-arrested NRK, human lung, and Swiss mouse 3T3 fibroblast monolayers, this effect being half-maximal at 2-3 pM beta TGF for NRK cells. These results identify eTGF and beta TGF as the two synergistically acting factors responsible for the transforming action of culture fluids from FeSV-Fre cells.

Genital warts and cervical cancer. V. The tissue basis of colposcopic change.

Histologic differences between subclinical papillomaviral infection and cervical intraepithelial neoplasia are quantitative rather than qualitative. Both entities are characterized colposcopically by acetowhite epithelium and aberrant surface capillaries. This study correlates five new colposcopic signs (variations in contour, thickness, color, vascular patterns, and iodine staining) with 12 histologic signs of human papillomaviral infection and 12 features of premalignant change. Acetowhitening and capillary abnormalities in minor colposcopic lesions are attributable to human papillomaviral disturbance of cell growth and maturation, seen histologically as parabasal layer proliferation, papillomatosis, koilocytosis , and dyskeratosis. Alteration in cell ploidy is usually minimal. Major colposcopic abnormalities reflect extensive disorganization of cell phenotype and tissue architecture, increased DNA content, and aneuploid stem cell populations. Intermediate colposcopic patterns generally denote polyploid lesions in which tissue changes are a composite of two reciprocal events: the extent of benign warty expression and the severity of premalignant change.

Polyoma virus transformation and two-dimensional electrophoresis patterns of baby hamster kidney cell native proteins.

The protein extracts of baby hamster kidney cells (BHK 21/C13) before and after malignant transformation by polyoma virus were resolved by isoelectric focusing in the first dimension and electrophoresis on concave 4-24% (w/v) polyacrylamide gradient gels in the second dimension. The resulting two-dimensional patterns were visualised by ultrasensitive silver staining. Major differences were observed in the protein distribution patterns of the two cell lines. Brief trypsinisation of the cells, prior to extraction of cell protein, had little effect upon the protein patterns of BHK cells. Similar treatment of the transformed cells markedly reduced the staining intensity of a string of spots which were present in far greater abundance than in the original BHK cells. The findings suggest that these proteins are located on the surface of the transformed cells and probably under the surface prior to transformation.

Changes in nucleolar and ribosomal RNA of the frog kidney after malignant transformation by the Lucke tumor herpesvirus.

Malignant transformation of the frog kidney by the Lucke herpesvirus changes the nucleotide base composition of normal kidney nucleolar and ribosomal RNA. In the Lucke tumor there is a moderate decline in guanylic acid and a sharp decline in adenylic acid levels. Conversely, there is a sharp increase in cytidylic acid and uridylic acid levels in the tumor cells. However, there was an increase in the G + C content of nucleolar and ribosomal RNA over that obtained from the normal kidney cells. Nearly identical quantitative changes in the base composition of each RNA species were measured for the adult (spontaneous) mesonephric carcinoma and a Lucke-herpesvirus-induced pronephric tumor cell line; similar correspondence was obtained for the normal adult mesonephros and a normal pronephric cell line.

Viral DNA integration patterns of primary SV40 tumors.

Primary tumors were induced in newborn Syrian hamsters by Simian virus 40 (SV40). These tumors were subjected to restriction enzyme analysis and each was found to have a unique viral integration pattern. One primary tumor was able to produce tumor foci in the lungs. Transplantation of this tumor resulted in a simplification of the integration pattern. The primary tumor was then cloned, revealing a heterogeneous population. When the cloned populations were transplanted subcutaneously into adult animals, restriction enzyme analysis of the tumors revealed no rearrangement of the viral DNA in either subcutaneous tumor or lung foci. Tumor cells isolated from the lungs were transplanted subcutaneously in adult animals and restriction enzyme analysis revealed an increase of free viral DNA and new sites of viral integration.

Using tropomyosin polymorphic phenomenon for detection of differences between rat spontaneous tumor cells and their in vitro oncovirus supertransformats.

Biochemical and immunological comparative studies of rat tumor cells of spontaneous origin and in vitro supertransformed cell populations have been done. We have focused on characterization of differences in tropomyosin molecular forms in the individual cell populations. Our experiments have shown that differences of tropomyosin forms exist not only between spontaneous transformants and supertransformants but also between supertransformants and normal rat fibroblasts. It means that superinfection of spontaneous transformants by avian sarcoma virus B77 have induced changes in tropomyosin synthesis but a pattern of tropomyosin forms in super-transformants has not been equal or similar to that of normal fibroblasts.