Human papillomavirus DNA in invasive carcinoma of the vagina.

The presence of human papillomavirus (HPV) DNA in invasive carcinomas of the vagina, in their lymph node metastases, and in corresponding normal tissue was investigated by Southern blot hybridization with 32P-labeled HPV DNA. Tumor tissue from ten of 18 patients with vaginal carcinoma contained HPV DNA. Three of the 18 patients had a history of cervical neoplasia more than 14 years before the diagnosis of vaginal carcinoma. Five of 15 primary squamous cell carcinomas, one primary adenocarcinoma, and a vulvar recurrence of a vaginal squamous carcinoma contained HPV 16. A primary squamous carcinoma yielded HPV-related sequences. The HPV copy number varied from 0.5 to 50 per cellular genome. Four histologically positive inguinal lymph nodes from three patients contained HPV DNA. In six tumor-free control tissues from four patients, no HPV DNA was detected. No relationship was established between HPV positivity, HPV type, or copy number of the tumor and the grade of differentiation or keratinization or the clinical stage. After a median follow-up of 13 months, five of nine HPV-positive patients were alive without recurrence, whereas all seven HPV-negative patients had died because of disease. The results of this study indicate a possible major role of HPV in the development of vaginal cancer.

Expression of epidermal growth factor receptor and HER-2/neu in normal and neoplastic cervix, vulva, and vagina.

Monoclonal antibodies were used to localize immunohistochemically epidermal growth factor receptor and HER-2/neu in normal and neoplastic frozen tissue samples from the lower genital tract of women. In squamous epithelia of the cervix, vulva, and vagina, epidermal growth factor receptor and HER-2/neu both were expressed most strongly by basal keratinocytes. Expression of both of these cell surface molecules decreased as cells underwent differentiation toward the mucosal surface. In contrast, both epidermal growth factor receptor and HER-2/neu were expressed throughout the entire thickness of the epithelium by undifferentiated squamous cells in squamous metaplasia, raised condyloma, and carcinoma in situ. In 34 squamous cancers of the cervix, vulva, and vagina, all malignant cells were found to have moderate to heavy staining for epidermal growth factor receptor. Staining of 33 of these cancers for HER-2/neu was light, although one patient who presented with distant metastases had heavy staining for HER-2/neu. These data suggest that although overexpression of HER-2/neu in squamous cancers of the lower genital tract is a rare event, it may be associated with aggressive biologic behavior.

Nuclear and "cytoplasmic" estrogen and progesterone receptors in squamous cell carcinoma of the cervix.

Biopsies from 131 women with squamous cell carcinoma of the cervix diagnosed between May 1983 and July 1986 were assayed for nuclear and "cytoplasmic" estrogen receptors (NER and CER). Progesterone receptors (PR) were also assayed in 45 of these tumors. About a third of the tumors contained both CER and NER. One or the other fraction contained ER in 76.9% of cases and PR in 66.6%. Although there was a trend for those women whose tumors contained CER or NER to have a better prognosis, this was not significant. There was no evidence that PR status affected the prognosis.

Keratin subtypes in carcinomas of the uterine cervix: implications for histogenesis and differential diagnosis.

Normal epithelia and carcinomas of the human uterine cervix were studied by monoclonal antibodies chain specific for cytokeratins 4, 8, 10, 13, 14, 18, and 19. Most cells in 13 examined squamous carcinomas revealed a cytokeratin phenotype detected in ectocervical basal cells and endocervical subcolumnar reserve cells: 8+, 14+, 18+, 19+, 4-, 10-, 13-. We propose that these two cell types are closely related or identical and that squamous carcinoma of the cervix originates in this cell type. In more differentiated tumor cells cytokeratins 4, 10, and 13, which are present in suprabasal layers of the normal ectocervical epithelium, were coexpressed with basal cell cytokeratins. Thus, contrary to previous beliefs, all cytokeratins detected in carcinomas were also present in normal epithelium of uterine cervix. The cytokeratin profile of cervical adenocarcinomas corresponded to that of columnar endocervical cells (8+, 18+, 19+), although two of the three adenocarcinomas also expressed cytokeratin 4, which in the normal endocervix was detected in scattered single columnar cells only. The new monoclonal antibody DE-K14, specific for cytokeratin 14, proved a specific marker of subcolumnar reserve cells in the endocervix. It was also the only one that reacted with all cervical squamous carcinomas but with none of the cervical adenocarcinomas and, as such, has a potential value for pathological differential diagnosis of cervical tumors.

Recognition of N-glycosidic carbohydrates on esophageal carcinoma cells by macrophage cell line THP-1.

Cell-to-cell contact between macrophages and tumor cells is an important initial reaction in a host defense mechanism against tumor cells. The authors have studied cell surface components of human esophageal carcinoma cells recognized by macrophages. Superoxide release from THP-1 cells, a human macrophage cell line, was analyzed in their interaction with a battery of human squamous cell carcinoma cell lines (TE) originated from esophageal cancer patients. The macrophage-triggering ability of TE 1 cell line, a high stimulant, was reduced after treatment with trypsin or tunicamycin, an inhibitor of N-glycosidic glycosylation. Addition of monosaccharides was efficient in competitive inhibition of these cellular interaction. Moreover, con-A-resistant mutation of TE 1 cells was found to reduce their macrophage-triggering ability, associated with increase of L-PHA-binding capacity, suggesting substitution to the GlcNAc beta(1----6)-linked lactosamine antenna in N-glycosidic carbohydrates. These findings suggest that terminal residues of N-glycosidic carbohydrates on some esophageal carcinoma cells may contribute to the recognition sites of macrophages.

Secreted phosphoprotein mRNA is induced during multi-stage carcinogenesis in mouse skin and correlates with the metastatic potential of murine fibroblasts.

Secreted phosphoprotein I (SPP), also known as 2ar, osteopontin, 44-kDa bone phosphoprotein, bone sialoprotein I, and transformation-related phosphoprotein, is a 41.5-kDa glycosylated phosphoprotein secreted by many mammalian cell lines and expressed in a limited set of tissues. Using a cDNA probe, we found that SPP mRNA, which is barely detectable in normal mouse epidermis, was expressed at moderate-to-high levels in 2 of 3 epidermal papillomas and at consistently high levels in 7 of 7 squamous-cell carcinomas induced by an initiation-promotion regimen. This contrasts with the transient induction we had previously observed after a single application of the tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA). In a set of 5 independently isolated T24-H-ras-transfected mouse C3H 10T1/2 cell lines, the levels of SPP mRNA correlated well with ras mRNA levels and with both experimental and spontaneous metastatic ability. SPP mRNA expression was also elevated in a derivative of mouse LTA cells transfected with genomic DNA from B16F1 melanoma cells and selected for increased experimental metastatic ability in the chick embryo. This apparent association of SPP expression with invasion, progression and metastasis, along with the presence of a functional ArgGlyAsp (RGD) cell adhesion site in SPP (osteopontin), leads us to propose that SPP may act as an autocrine adhesion factor for tumor cells.

Expression of ras oncogene p21 protein in esophageal squamous cell carcinoma.

In order to investigate the value of ras oncogene expression as a prognostic indicator in esophageal squamous cell carcinoma, we evaluated the level of ras oncogene protein product (p21) in 52 specimens resected between 1977 and 1986. All patients were followed until death or for at least 2 years. Pathology slides and archival paraffin blocks were retrieved for confirmation of the original diagnosis, study of histopathologic features, and measurement of p21 content. P21 titers were obtained using the RAP-5 monoclonal antibody in a semiquantitative immunohistochemical assay. Titer was expressed as the highest dilution of antibody giving definitive staining using the avidin-biotin peroxidase method. Ras oncogene was expressed in 88.5% of the specimens. We did not find a significant correlation between ras expression and any of a variety of clinical and histopathologic prognostic parameters. Although patients' median survival after resection of specimens with ras oncogene expression was less than half the median survival after removal of tumors without such expression, this difference was not statistically significant. Further prospective investigations are needed to assess the role of ras oncogene evaluation in clinical practice.

Coexpression of cytokeratins, involucrin, and blood group antigens in oral squamous cell carcinomas.

The well and poorly differentiated oral squamous carcinomas preferentially express proteins, blood group antigens, and contain associated dendritic Langerhans' cells. Keratin pearls in well-differentiated carcinomas simulate the differentiation pathway of the normal oral squamous epithelium, whereas poorly differentiated carcinomas do not and appear more heterogeneous. Terminally keratinized cells correlate with involucrin and expression of blood group antigens in keratin pearls, a feature that differs from the nonkeratinizing normal epithelium in which such carcinomas arise. Dendritic Langerhans' cells are reduced in number in squamous carcinomas.

[Analysis of cellular DNA-RNA content using flow cytometry between primary and metastatic foci in lung carcinoma].

By flow cytometry, cellular DNA-RNA content was analyzed with Acridine Orange staining for primary tumor and the metastatic foci in 12 cases of primary lung carcinoma. Five (42%) of the primary and metastatic foci showed heterogeneous cellular DNA content, with high cellular DNA content of metastatic foci in all cases. Cellular RNA content was significantly higher in metastatic foci than in primary foci, indicating possible higher proliferative activity of the former foci. Three different metastatic foci in one patient showed essentially the same cellular DNA content. From the above it is evident that primary and metastatic foci of lung carcinoma show considerable heterogeneity not only in cellular DNA content but RNA content as well, thus indicating proliferative activity of the metastatic focus. It is consequently advisable to use a combination of various supplementary medications differing in cytocidal effect. Cases with higher cellular DNA content may likely have higher metastatic potential.

Whole cell preparation of squamous cell carcinoma for cytometric analysis of DNA.

To date, analysis of the DNA content of head and neck squamous cell carcinomas has relied on the homogenation of the entire tissue specimen and subsequent staining and quantitation of the naked nuclei. This methodology does not make allowance for the extremely variable nature of these tumors with respect to their cellular composition. Further, by destroying the cytoplasm and cell membranes, this methodology makes it impossible to distinguish the DNA content of the tumor cells from that of the background stromal and inflammatory cells. The authors present a methodology for the selective exclusion of inflammatory cell infiltrates from the DNA analysis of these tumors. Using this technique, it has been found that exclusion of the inflammatory cells allows the investigator to look more specifically at the malignant cell population. This has been most helpful in those samples in which the tumor cells have been diploid or near-diploid. With this technical refinement, the relationship between DNA ploidy and clinical prognosis may be more accurately assessed.

An immunohistochemical assessment of cellular proliferation markers in head and neck squamous cell cancers.

Prognostic information is essential for the evaluation, judgement and optimal treatment of patients with squamous cell cancers (SCCs) of the upper aerodigestive tract. Using immunohistochemical and flow cytometric techniques, we have studied the significance of cellular expression of the Ki-67 antigen, epidermal growth factor receptor (EGFR), the transferrin receptor (TFR) and DNA ploidy status in a prospective analysis of patients with SCCs of the head and neck region. All 42 fresh tumour samples (five well differentiated; 28 moderately differentiated; nine poorly differentiated) expressed both EGFR and TFR to varying degrees. Receptor expression was most marked on the peripheral invading margin of cancer cell islands although staining was also demonstrated in a random fashion within cellular islands and consistently along the basal cell layer of overlying stratified squamous epithelium. The percentage of cancer cells that reacted with the Ki-67 monoclonal antibody was assessed as low (less than 10%) in 15 samples (35.8%), intermediate (10-30%) in 19 samples (45.2%) and high (greater than 30%) in eight samples (19.0%). Eleven of 15 samples (73%) with a low percentage reactivity were DNA diploid, whereas seven of eight samples (87.5%) with a high percentage reactivity were DNA aneuploid. Poorly differentiated SCCs were significantly more often aneuploid than were either moderately or well differentiated tumours. Our results suggest that EGFR and TFR are widely distributed on SCCs, especially on proliferating cells at the invading tumour margin. In addition, there is a close spatial correlation between cells expressing EGFR, TFR and those expressing the Ki-67 antigen. Tumours in which the staining intensity for both EGFR and TFR was intense invariably expressed the Ki-67 antigen in a high proportion of cells. Further patient follow-up will be important in determining whether intense EGFR and TFR staining, combined with a high percentage reactivity with Ki-67 antibody and DNA aneuploidy, will ultimately define a subset of head and neck cancer patients with a poor clinical outcome.

[Evaluation of double cancers in relation to previous asbestos exposure].

Ten cases of double cancers (a lung cancer and a stomach cancer) were evaluated in relation to previous asbestos exposure. Ten cases involved male more than 69 years old. Five cases had developed their two cancers simultaneously and other 5 cases had developed their lung cancer after stomach cancer surgery. The lung cancer was their main disease. Four cases had early stage stomach cancers and 5 cases with a stomach cancer had no relapse after surgery. Eight cases had occupational histories of asbestos exposure. Significantly high numbers of asbestos bodies were detected in the autopsied lung in almost all cases. According to the X-ray analysis, almost all asbestos fibers detected in the lung were chrysotile. Additionally, the Brinkman Index (B.I.) of these 10 cases ranged between 700 and 2,000. The combination of asbestos exposure and smoking is thought to be an important factor in the development of such double cancers.

[Immunohistological coexpression of keratin and vimentin in the epithelial neoplastic cells].

Filed formalin-fixed paraffin blocks of 128 cases of epithelial neoplasms were selected for immunohistochemical study of keratin and vimentin expression. The results showed that 35.1% (45/128) of different carcinomas expressed vimentin. The immuno-positivity of vimentin in thyroid carcinomas, ovarian carcinomas, prostatic adenocarcinomas, pulmonary carcinomas and malignant mesotheliomas were 81.8%, 42.8%, 66.7%, 30.5% and 53.4%, respectively. Carcinomas of breast, kidneys, salivary glands, adrenal glands and nasopharyngeal carcinomas also showed various degrees of positive reaction. The results suggest that an immunohistochemical positive vimentin reaction does not exclude histopathological diagnosis of carcinomas. The significance and noticeable aspects of immunohistochemical methods in histopathological diagnosis are also discussed.

Cytokinetic investigation of lung tumors using the anti-bromodeoxyuridine (BUdR) monoclonal antibody method: comparison with DNA flow cytometric data.

In order to investigate the cytokinetics of malignant tumors and non-malignant lesions of the lung, tissue samples from 57 patients affected by non-small-cell carcinoma (NSCLC), small-cell carcinoma (SCLC), and benign and inflammatory lesions have been analyzed using the BUdR monoclonal antibody (MAb) method. This method is based on the preparation, at the time of surgery, of viable monocellular suspensions (using collagenase and DNase treatment) and the concomitant administration of BudR. The percentage of BudR-labelled cells was monitored by fluorescent microscopy using an FITC-labelled second antibody. In NSCLC, each histological group showed a wide range of labelling index (LI) values. On the contrary, SCLC exhibited a more homogeneous kinetic behaviour as evidenced by a narrowly distributed, higher LI. Tumors shown to be diploid by flow cytometry did not show a lower LI than aneuploid tumors. Furthermore, differences were constantly observed between the S-phase percent calculated using BUdR and that calculated using the DNA flow cytometric (FC) histogram, the latter always showing higher S-phase values. In an attempt to study the intra-tumor proliferative heterogeneity, multiple-site sampling was performed. Proliferative heterogeneity seemed to be higher inter-tumor than intra-tumor. Finally, a positive correlation (p less than 0.05) was found between LI and the actual doubling time (DT) of the primary tumor mass, evaluated using sequential radiographs. In conclusion, the present BUdR method can be considered a useful source of relevant information on in vivo cell growth, in parallel to other clinical (DT) and biological (DNA content) approaches.

Distinct patterns of chromogranin A-related species can be demonstrated in endocrine cells.

We have studied the pattern of chromogranin A (CgA)-related species in different human endocrine cells that produce CgA and also express the calcitonin gene. Antibodies against CgA peptides that span its linear sequence were used in Western analysis of cell lines derived from medullary thyroid carcinoma (MTC), small cell lung cancers (SCLC), epidermoid cell lung cancer (ECLC) and a pulmonary carcinoid tumor (CRND). Each of the cell lines demonstrated a distinct pattern of CgA-related species. Gel filtration studies also revealed multiple and different forms of immunoreactive CgA in the cell lines. Although proteolysis may contribute to our results, these observations suggest that native CgA is processed to smaller species in a tissue-specific pattern by different endocrine cells. More conclusive studies, however, are necessary to establish that cell processing leads to the specific CgA moieties that we have observed.

Esophageal carcinoma-associated proteins detected by two-dimensional polyacrylamide gel electrophoresis.

Patterns of proteins of five surgically resected esophageal carcinomas were studied by two-dimensional polyacrylamide gel electrophoresis with silver staining. The samples of normal esophageal mucosa and esophageal carcinoma from the same patient were compared. Each gel had ca. 300 protein spots and had a similar pattern of proteins. Four spots were observed in all of the esophageal carcinomas that were not present in any of the normal mucosae. The molecular weights and isoelectric points were 46,000 and 5.3, 46,000 and 5.2, 36,000 and 4.7 and 33,000 and 5.1, respectively. One spot was observed in all of the normal mucosae but not in any of the esophageal carcinomas. Its molecular weight and isoelectric point were 27,000 and 5.3, respectively.

[Studies on DNA content in ploidy patterns of esophageal cancer].

The present study was undertaken to evaluate the nuclear DNA content of esophageal cancer cells consequent on the process of growth and progression of the cancer. In experimental animal studies, 47 esophageal cancers were induced in male Wistar rats by oral administration of N-amyl-N-methylnitrosamine (AMN) and were analysed in the study of ploidy patterns. The study was also carried out to determine the DNA content in the ploidy patterns in man. Primary tumors associated 421 metastatic lymph nodes in the 62 patients with thoracic esophageal cancer were subjected for the study of ploidy patterns. The nuclear DNA content was determined by means of flow cytometry. In the study of the experimentally-induced esophageal cancer in rats, aneuploidy was found in 18% at a depth of submucosa, 30% at proprial muscle, 59% at adventitia, and in 50% at a depth of neighboring structures, respectively. Clinically in man, the incidence of DNA diploidy and aneuploidy in the 62 primary cancers was 56% and 44%, respectively. In the 421 metastatic lymph nodes, diploid was found in 73% and aneuploid in 11%, while the combination of diploid and aneuploid was observed in 16%. Difference in the DNA index (DI) between the primary cancers and metastatic lymph nodes was found in 29 cases (46.8%), and the difference increased with progression of the cancer. Two hundred and ninety seven metastatic lymph nodes of 29 cases were subdivided into 4 groups based on the extent of the cancerous nests, and the DI value was found to be increased in proportion to the extension. With the results, the DI value of esophageal cancer appeared to be changed dependently by the variation of cell populations in the cancer or in the DNA content of the cancer cells.

[Squamous cell carcinoma-associated antigen (SCC) as a tumor marker in the initial diagnosis of carcinomas of the head and neck region. Results of a prospective study after 24 months]. / Das plattenepithelkarzinomassoziierte Antigen (SCC) als Tumormarker bei der Initialdiagnostik von Karzinomen des Kopf-Hals-Gebietes. Ergebnisse einer prospektiven Studie nach 24 Monaten.

The serum--SCC antigen levels of patients with head and neck tumors were studied prospectively to determine their value in the initial diagnosis of head- and neck-cancer patients. Serum concentrations above 2 ng/ml are considered abnormal. Preliminary results of the study after a 12-month period have been reported elsewhere (1). The final results of the study show an increased percentage (53%) of pathologic findings, mostly due to the increasing number of advanced stage tumors. High serum levels were found in 60% of the T4-tumors (Fig. 4a). Well differentiated carcinomas seem to be associated with the antigen more frequently than poorly differentiated tumors (Fig. 5). SCC antigen levels were examined as many as five times before the start of treatment (85 patients), and in one-third of those cases the differences between the serum levels exceeded 1 ng/ml. As far as 85% specificity is concerned, the ROC-curve shows a sensitivity of only 40% (Fig. 2) which, in addition to the fact that the antigen was most frequently found in cases of advanced tumors, indicates that the usefulness of the SCC antigen as a tumor marker for head and neck cancer must still be regarded as low.

Association between poor prognosis in early-stage invasive cervical carcinomas and non-detection of HPV DNA.

Human papilloma virus (HPV) DNA sequences (HPV types 16, 18, 33, 35 or uncharacterized) were detected by Southern blot hybridisation and polymerase chain reaction in 84% of 106 early-stage invasive carcinomas of the uterine cervix. Among HPV-positive patients, the risk of overall relapse did not differ with individual HPV types. Compared with HPV-positive patients, those with no detectable HPV DNA had a 2.6 times higher risk of overall relapse (p less than 0.05) and 4.5 times higher risk of distant metastases (p less than 0.01). The 24-month relapse-free survival rate in HPV-positive patients was significantly higher than that in HPV-negative patients (77% vs 40%), and the difference was similar (91% vs 56%) among those who were node-negative. These data indicate that HPV-negative cervical carcinomas may represent a biologically distinct subset of tumours that carry a poorer prognosis than do HPV-positive cancers.