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.

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.

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.

Glutathione and cysteine levels in human tumour biopsies.

There is evidence that some human tumours could be treated with a combination of buthionine sulfoximine and hypoxic cell sensitizers. However, clinical application of this technique requires a prior knowledge of the level of non-protein bound sulfhydryl (NPSH) compounds in these tumours. The present study provides data on the levels of glutathione (GSH) and cysteine (CYS) in human tumour biopsies from the cervix and from the head and neck. The NPSH compounds were measured by high performance liquid chromatography. The median GSH values were 20.5 nmol/mg protein (cervix) and 23 nmol/mg protein (head and neck) while the median CYS values were 4.4 (cervix) and 4.2 nmol/mg protein (head and neck). The values varied widely from one patient to another.

Human papillomavirus infection of the uterine cervix analyzed by nonisotopic in situ hybridization.

Some types of human papillomaviruses (HPVs) have been suggested to be strongly related to uterine cervical carcinoma. An attempt to detect these in formalin-fixed, paraffin-embedded sections was made by either immunohistochemical or by in situ hybridization. Anticapsid protein of bovine papillomavirus antibody labeled with peroxidase was used for immunohistochemistry, and biotin was used instead of radioisotopes to label probes for in situ hybridization, which resulted in low background and a rapid procedure. Condylomatous changes were stained immunochemically with this antibody even in invasive carcinoma, whereas the carcinoma itself was not stained. Direct correlation was demonstrated by in situ hybridization between the HPV genome and histopathological structure, which was impossible by Southern or dot hybridization. HPV DNAs were detected in the nuclei of koilocytes and dyskeratinocytes of condylomata and dysplasias. Furthermore, hybridization signals of HPV DNAs in basal and parabasal cells suggested that HPV infection had already begun in the basal cells. In the case of malignant neoplasia accompanied by dysplasia, the same type of HPV was detected both in the malignant neoplasia and accompanying dysplasia. In one case of intraepithelial carcinoma, the very small focus of carcinoma just arisen in the cells of dysplasia was identified, and both were positive for HPV 18. This fact supports the suggestion that the carcinoma arises in dysplasia. Invasive carcinomas were classified further into keratinized, large-cell nonkeratinized, and small-cell nonkeratinized types, and the positive frequency for HPV 16 decreased as the differentiation of the carcinoma decreased. In the case of keratinized type of invasive carcinoma, strong hybridization signals were prominent around the malignant pearl formation.

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.

Immunoreactive alpha interferon in cervical flat koilocytic lesions and intraepithelial neoplasia.

Immunocytochemical staining for alpha-interferon was carried out on cervical biopsy specimens showing non-condylomatous koilocytic atypia (n = 12) and cervical intraepithelial neoplasia (n = 18), both of which are associated with human papilloma virus (HPV) infection. Normal cervical tissue obtained from hysterectomy specimens was also assessed. Koilocytes were not immunoreactive for alpha interferon and keratinocyte staining was observed in only four cases of intraepithelial neoplasia. HPV infection alone does not therefore seem to induce the production of alpha interferon in cervical squamous epithelium. There was variable but, in some cases, prominent staining of cells in the stromal inflammatory infiltrate as well as intraepithelial cells which had morphological and immunocytochemical characteristics of Langerhans' cells. Alpha interferon immunoreactivity in Langerhans' cells is in keeping with derivation from the mononuclear phagocyte system and may be important in the host response to HPV infection.

Temporal associations of human papillomavirus infection with cervical cytologic abnormalities.

The relationship between infection with different human papillomavirus types and cervical intraepithelial neoplasia was studied in a group of 398 women seen in a private gynecology practice in Washington, D.C. Each woman was assessed for human papillomavirus infection by Southern blot hybridization analysis of cervical cells obtained by swab. The human papillomavirus results were correlated with the results of Papanicolaou smears taken the same day and with data abstracted from medical records regarding past cervical disease. Subjects with normal cytologic findings at the time of human papillomavirus testing were followed up for an average of 2 to 3 years with additional Papanicolaou smears. At the time of human papillomavirus testing, 58% (19/33) of women with cervical intraepithelial neoplasia had detectable human papillomavirus deoxyribonucleic acid in contrast to 10% (28/289) of women with normal cytologic findings (p less than 0.001). This association persisted after statistical adjustment for age and current use of oral contraceptives, a factor that appeared to increase the detection of human papillomavirus. Among women with no current cytologic evidence of neoplasia, human papillomavirus detection was more likely in those with a history of past genital neoplasia (p = 0.05). In the follow-up study, 15% (3 of 20) of cytologically normal women who were human papillomavirus-positive at baseline subsequently exhibited cervical cells suggestive of cervical intraepithelial neoplasia compared with only 5% (9 of 195) of human papillomavirus-negative women. However, this difference reflected recurrent and not incident neoplasia.

Enhanced chemiluminescence for tissue antigen and cellular viral DNA detection.

The ability to use enhanced chemiluminescence (ECL) to detect horseradish peroxidase as a label for tissue antigens and cellular viral DNA was demonstrated. A liquid nitrogen-cooled charged-coupled device (CCD) was used to detect light output, which was visualized on a monitor or was quantitated using an attached microcomputer. In a tissue antigen model, equivalent sensitivity was observed between ECL and colorimetric detection.

Mapping of linear epitopes of human papillomavirus type 16: the L1 and L2 open reading frames.

Certain types of human papillomavirus (HPV), notably HPV type 16, are associated with flat or inverted proliferative lesions of the cervix uteri that can progress to malignancy. As a first step towards the serological study of the epidemiology of HPV, we have synthesized the entire amino acid sequences of the 2 major viral capsid proteins of HPV type 16, L1 and L2, as a set of 66 synthetic 20-residue peptides with an overlap of 5 amino acids. The peptides were tested for reactivity with IgA, IgG and IgM antibodies in the sera of 30 patients with HPV-16-carrying cervical neoplasms. Both IgG and IgM antibody responses were detected, but most of the reactivity found was of the IgA class. The most immunoreactive peptides were further analyzed for reactivity with sera from 22 patients with parotid gland tumors and with sera from 38 healthy individuals. The L2-encoded protein contained only one major linear epitope, which was not specific for HPV-16-carrying neoplasms. In contrast, the L1-encoded protein contained several epitopes that were regularly immunoreactive with antibodies present in the sera of patients with HPV-16-carrying cervical neoplasms, but only rarely so in the sera of patients with other tumors or of healthy individuals.

Detection of pS2 messenger RNA in gynecological cancers.

Estrogen-inducible pS2 mRNA was previously detected in human cancer cell lines the growth of which was sensitive to estrogen. In the present study, the expression of the pS2 gene was analyzed in 111 gynecological carcinomas. The pS2 message was detected in greatest abundance in 6 primary carcinomas of the ovary (6 of 29), 4 of these being mucinous cystadenocarcinomas. A secondary carcinoma of the ovary, and another of the omentum (1 of 4), also contained detectable levels of pS2 mRNA. Weak pS2 mRNA signals were occasionally observed in endometrial (2 of 55) and cervical carcinomas (2 of 33) as well. There was a poor correlation between estrogen receptor and pS2 mRNA in ovarian carcinomas.

In situ hybridization analysis of HPV DNA in cervical precancer and cervical cancers from China.

A series of 103 cervical biopsies derived from 103 women during July 1958 to September 1963 from Beijing, China were investigated with in situ hybridization for the presence of HPV6, 11, 16, 18, 31 and 33 DNA. The mean age of the patients was 46.1 + 10.6 years with a range of 24-74 years. Morphological features of HPV infection were found in 80 (77.7%) biopsies. Invasive cervical cancer was diagnosed in 43 biopsies and cervical intraepithelial neoplasia CIN I, CIN II and CIN III in 9, 9, and 27 cases, respectively. A total of 63.1% (65/103) of the lesions had morphological features of HPV infections associated with CIN or invasive carcinomas. Altogether, 31.1% (32/103) of the biopsies were shown to contain HPV DNA. Of the cases showing HPV morphology, 43.1% were HPV DNA positive. HPV16 (30/32) was the most frequent type, followed by HPV11 and 18, whereas no lesions with HPV6, 31 or 33 were found. A total of 19/43 (44.2%) of the invasive carcinomas contained HPV DNA. HPV DNA positivity and the grade of CIN showed a statistically significant correlation (P = 0.0011). Our study demonstrated the presence of HPV in cervical lesions among Chinese women in the late 1950's and early 1960's when a single sexual partner was the rule and also supports the concept that HPV has as an important etiological role in cervical cancer, the highest risk being associated with HPV type 16. The applicability of in situ hybridization in retrospective assessment is emphasized.

Minimal deviation adenocarcinoma (adenoma malignum) of the endocervix: a histochemical and immunohistochemical study of two cases.

The histopathological diagnosis of minimal deviation adenocarcinoma (adenoma malignum) of the endocervix may be difficult. Two cases of minimal deviation adenocarcinoma (MDA) were examined using mucin histochemistry and immunocytochemistry with antibodies to epithelial membrane antigens (HMFG1, Ep1), low-molecular-weight cytokeratins (CAM 5.2), carcinoembryonic antigen (CEA), and alpha-amylase. The results were compared with those for normal endocervical glands. Reactivity for CEA in MDA was focal and would be unreliable for biopsy diagnosis. Both cases of MDA contained abundant neutral mucins and sialomucins, whereas sulfomucins were rarely detected; this pattern contrasted with that of normal endocervix. Neoplastic glandular epithelial cells in MDA consistently showed both luminal and cytoplasmic reactivity with Ep1 and HMFG1, whereas normal cervix showed luminal labeling only. Thus, mucin histochemistry and immunohistochemical detection of epithelial membrane antigens may distinguish between extremely well differentiated neoplastic glands in MDA and normal endocervical glands, and hence may aid diagnosis in biopsy specimens.

MSN-1 antibody in the evaluation of female genital tract adenocarcinomas.

The reactivity of a new monoclonal antibody (MAb), MSN-1, raised against a human endometrial cancer cell line (SNG-II), was studied in a variety of endometrial, endocervical, and ovarian carcinomas as well as normal cycling endometrium. Moderate to strong reactivity (2-3+) was seen in six of nine normal secretory endometria (67%), one of 10 normal proliferative endometria (10%), 18 of 18 endometrial adenocarcinomas (100%), 10 of 11 endometrioid ovarian adenocarcinomas (91%), seven of nine clear cell ovarian adenocarcinomas (78%), one of 12 endometrial hyperplasias without atypia (9%), two of four endometrial hyperplasias with atypia (50%), zero of five endometrial serous adenocarcinomas, two of 17 serous ovarian adenocarcinomas (12%), zero of 10 intestinal-type mucinous ovarian adenocarcinomas, and zero of nine metastatic adenocarcinomas in ovary. Endocervical adenocarcinomas showed moderate to strong staining in 75% (six of eight). It is concluded that MSN-1 can be used to confirm endometrioid/clear cell differentiation in ovarian and endometrial tumors, cannot be used to discriminate endocervical from endometrial differentiation, cannot be used to discriminate atypical hyperplasia from carcinoma, and may be useful to distinguish between atypical (premalignant) endometrial hyperplasias and those without atypia.

Ultrastructural and immunohistochemical study of infiltration in microinvasive carcinoma of the uterine cervix.

Carcinoma in situ and microinvasive cancer of the cervix were compared by transmission electron microscopy to examine ultrastructural features of the locally infiltrating lesion of microinvasive cancer. Many pseudopod-like cytoplasmic protrusions of the cancer cells and abundant microfilaments parallel to the direction of the protrusion were seen. Concomitant with the disappearance of part of the basal lamina, many vesicles 70-90 nm in diameter were observed, suggesting a role for these vesicles in cancer infiltration. With the immunoperoxidase method, the distribution of fibronectin around the invasive lesion also was examined. Fibronectin is a component of extracellular matrices and presumably, in view of its action on cell adhesion, is a resistant factor against cancer cell infiltration. Fibronectin decreased in the transitional area between the cancer nest and the stroma during the stage of microinvasion.

Distribution of involucrin in normal and pathological human uterine cervix.

A study was undertaken to determine the potential value of involucrin immunostaining, a protein synthesized by mature squamous epithelial cells, in distinguishing benign from neoplastic lesions in cervical pathology. A total of 146 cervical biopsies were analyzed using an indirect immunoperoxidase method and polyclonal antibody. A suprabasal homogeneous cytoplasmic staining pattern was consistently observed in normal squamous cervical epithelium. In contrast, 43.7% of cervical condylomas showed involucrin at all levels of the epithelium including the basal layer. Variable patterns were seen in cervical intraepithelial neoplasia (CIN), with 46% of full-thickness stainings, although no significant difference was obtained among the different grades of CIN lesions. Distribution of involucrin was correlated (P less than 0.05) with the degree of tumor differentiation in squamous cell carcinomas, being absent in 71.4% of poorly differentiated carcinomas and focally present in 75% of well-differentiated carcinoma. Lesions of endocervical origin, either benign or malignant, were entirely negative for involucrin. It is concluded that involucrin seems unable to establish a reliable differential diagnosis between benign and neoplastic conditions in cervical pathology, and should therefore be considered only a specific marker of squamous differentiation in both normal and pathological human uterine cervix.

Human papillomavirus associated with adenocarcinoma and adenosquamous carcinoma of the cervix: analysis by in situ hybridization.

The incidence of adenocarcinoma of the cervix appears to be increasing. Recent reports have demonstrated an association between adenocarcinoma of the cervix and human papillomavirus (HPV) by Southern blot hybridizations. In situ deoxyribonucleic acid (DNA) hybridization was performed on paraffin-embedded specimens to localize the source of HPV DNA. In pure adenocarcinoma five of six specimens were positive for HPV DNA. Four specimens contained HPV type 18 and one HPV type 16. Only one of three adenosquamous lesions was positive and it contained both HPV types 16 and 31. These findings suggest an association between HPV and adenocarcinoma of the cervix.

The expression of EGF receptors, EGF-like factors and c-myc in ovarian and cervical carcinomas and their potential clinical significance.

The expression of the epidermal growth factor receptor (EGF-R) and the c-myc oncogene was investigated in different specimens of ovarian and cervical carcinomas. The EGF-Rs were analyzed by EGF binding assay, immunohistochemistry and Northern blotting. For analysis of c-myc expression, we used Northern blotting and immunohistochemistry. Furthermore, tissue concentrations of EGF-like factors (EGF-F) were measured in the same tumor and non-malignant specimens. The biochemical determination of EGF-R demonstrated that EGF specific binding sites were detected in 36% of ovarian (n = 140) and 81% of cervical carcinomas (n = 42). High amounts of EGF-R (greater than 10 fmol/mg specific binding) were found in 8% of the ovarian and 41% of the cervical carcinomas. Increased expression of EGF-R specific mRNA was detectable in 7/21 ovarian and in 5/7 cervical carcinomas. A positive correlation between the amounts of EGF-R mRNA, the EGF-R binding data and the staining index of EGF-R immunohistochemistry was found. The EGF-R immunohistochemistry demonstrates that only the tumor cells produce increased amounts of EGF-R, while the stromal cells are EGF-R negative. Low amounts of EGF-R specific mRNA were also detected in biochemically EGF-R negative tumors. The c-myc specific mRNA signal was found in all cases investigated. It is shown that the c-myc expression was increased in 10/21 ovarian and 5/7 cervical carcinomas. There was no positive correlation between the amounts of EGF-R and c-myc mRNAs. The product of myc, as detected by immunohistochemistry, is found in tumor as well as in stromal cells. The levels of EGF-F were measured in extracts of 63 ovarian and 12 cervical carcinomas and in 21 non-malignant tissues. About 30% of the tumor extracts contained higher EGF-F levels (4-15 ng/mg) than those found in the non-malignant specimens. Tumors with high EGF-F levels expressed high amounts of c-myc RNA. The EGF-R status (n = 111) and the EGF-F levels (n = 63) were related to the prognosis of survival for patients with ovarian carcinomas. EGF-R positive (EGF-R(+)) ovarian carcinomas had a significantly higher response rate to chemotherapy. The survival time of the EGF-R(+) group is reduced compared to the EGF-R negative (EGF-R(-)) group if only patients in remission are used to construct survival curves. Furthermore, a poor prognosis for survival was noticed for ovarian carcinoma patients with high EGF-F levels.

[Detection of HPV-DNA in the various uterocervical lesions by in situ hybridization].

We detected HPV (Human Papilloma Virus) -DNA in various uterocervical lesions by in situ hybridization using biotinylated DNA probes. In cases positive for HPV, the nuclei of the epithelial cells was purple to blackish purple. In 2 of 6 cases of chronic cervicitis, HPV-DNA was detected in the outer layer of the squamous epithelium. Eleven of 19 with mild dysplasia (57.9%) showed a positive reaction in the upper one-third of the epidermis in a mainly consisting of koilocytotic cells. All 6 patients with moderate dysplasia had positive cells among the koilocytotic cells and atypical cells in the middle layer. Five of 11 patients with severe dysplasia had scattered positive cells. Two of them had atypical condylomatoid lesions. Eight of 32 patients with squamous cell carcinoma were positive for HPV-DNA, but there was no consistent distribution pattern of the positive cells.