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.

Human papillomavirus type 18 in conjunctival intraepithelial neoplasia.

Human papillomaviruses are oncogenic viruses that have been found in a variety of epithelial neoplasias. We sought to confirm their presence in conjunctival intraepithelial neoplasia. Five tumors were studied with a polymerase chain-reaction assay designed to detect the E6 region of human papillomavirus types 16 and 18. Human papillomavirus type-16 DNA was found in four of the five tumors, including two tumors that contained both type-16 and type-18 DNA. Viral DNA was not present in the fifth tumor.

Human papillomavirus DNA in genital tract lesions histologically negative for condylomata. Analysis by in situ, Southern blot hybridization and the polymerase chain reaction.

Koilocytotic atypia (nuclear atypia in conjunction with perinuclear halos) is diagnostic of condylomata of the lower female genital tract, over 90% of which contain human papillomavirus (HPV) DNA. Genital tract lesions may be clinically suggestive of condylomata but lack clear-cut koilocytotic atypia. Of 57 vulvar and 60 cervical lesions that lacked clear-cut koilocytotic atypia, four (7%) and two (3%), respectively, had HPV DNA detected by in situ analysis. Using Southern blot analysis, HPV DNA was detected in five of 27 (19%) and 20 of 55 (36%) vulvar and cervical lesions, respectively, that lacked koilocytotic atypia. When analyzed with the polymerase chain reaction (PCR), HPV DNA was detected in six of 22 (27%) and three of 18 (17%) vulvar and cervical lesions, respectively, that lacked koilocytotic atypia. These findings demonstrate that infection by HPV may be found in genital tract lesions that lack koilocytotic atypia. The lower detection rate of HPV in cervical lesions that lacked koilocytotic atypia with PCR as compared with Southern blot analysis may be related to the relatively high proportion of "novel" types (related to, but distinct from, the HPV types in the probe) in such lesions. The increase in the detection rate in vulvar lesions that lacked koilocytotic atypia with PCR compared with in situ hybridization suggests that about one third of such lesions are HPV related, but that in such cases the copy number of the virus is typically below the threshold of the in situ analysis.

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.

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.

Uterine adrenergic and cholinesterase-positive nerves and myometrial catecholamine concentrations during pregnancy in sheep.

Uterine adrenergic and cholinesterase (AChE)-positive innervation of the sheep uterus during anestrus and at 4 stages of pregnancy were examined by histochemical methods. In addition, uterine and cervical myometrium concentrations of norepinephrine (NE) and dopamine (DA) were determined using high-performance liquid chromatography. During anestrus, adrenergic and AChE-positive nerve fibers in the uterine myometrium and endometrium were primarily associated with the vasculature. Innervation of myometrial smooth muscle was almost exclusively by adrenergic fibers. In the endometrium, fibers of both types were observed closely associated with endometrial glands, and adrenergic fibers were observed in the connective tissue beneath the luminal epithelium. Density of uterine innervation decreased by day 65 of pregnancy with an additional decrease by day 105. Myometrial NE concentrations were higher in the cervix than the uterus. Uterine NE concentrations generally were not affected by pregnancy. Although cervical NE per gram of tissue decreased during pregnancy, this effect of pregnancy was not detected when NE was expressed per microgram of DNA. Myometrial DA concentrations were higher in uterine segments than in the cervix. DA concentrations decreased during pregnancy in all tissues except the posterior uterine segment. The DA to NE ratio in the uterus was greater than that for the cervix and was not generally affected by the stage of pregnancy. These results demonstrate that cholinergic and adrenergic nerves supply the sheep uterus. Decreasing fiber density during pregnancy suggests that a majority of the innervation to the sheep uterus is supplied by 'short' nerve fibers whose activity is regulated by steroids of pregnancy. The possible role of DA as a neurotransmitter in the sheep uterus is discussed.

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.

Relationship between dilatation of the rat uterine cervix and a small dermatan sulfate proteoglycan.

Cervical linear circumference (lo), extensibility and rate of creep, and the content and concentration of collagen and proteoglycans were determined on uterine cervices of rats at different reproductive stages. The inner circumference increased from 9 +/- 3 (SD) mm at the nongravid stage to a maximum of 41 +/- 5 mm at term; a significant drop to 23 +/- 2 mm occurred by 4 h postpartum with a further drop to 18 +/- 4 mm by 1 day postpartum. The extensibility and rate of creep reached their maxima 1 day before term and returned to the nongravid value by 1 day postpartum. The small (Mr = 95,000) type II dermatan sulfate proteoglycan, the major cervical proteoglycan, increased from 43 +/- 6 micrograms per cervix at the nongravid stage to 196 +/- 33 micrograms at term. The amount of this proteoglycan decreased significantly by 35% to 126 +/- 5 micrograms within 4 h postpartum and declined further to 79 +/- 16 micrograms by 1 day postpartum. The total cervical collagen content increased less than 2-fold during pregnancy, from 3.5 +/- 0.5 to 6.3 +/- 0.7 mg; a decline to 5.8 mg by 1 day postpartum was not significant. The ratio of small proteoglycan: collagen increased 2.5-fold between the nongravid state and term, then returned to the nongravid value by 1 day postpartum. Significant correlations were found between the lo and the amount of small proteoglycan per cervix (r = 0.86; n = 69) and between lo and the ratio of small proteoglycan:collagen (r = 0.83; n = 50) when data from every reproductive stage were combined. A mechanism is proposed whereby the interaction of the proteoglycan with collagen fibers might alter mechanical properties and contribute to cervical dilatation and its rapid reversal.

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.

S-100 protein in glands within decidua and cervical glands during early pregnancy.

Strong immunoreactivity with polyclonal S-100 protein antisera and monoclonal S-100 alpha subunit antiserum was found in glandular cells of the decidua basalis and cervical polyps during early pregnancy. Immunoreactive S-100 protein was negative in glandular cells of the endometrium and cervix of nonpregnant women. It was also negative in endometrial hyperplasia and endometrial carcinoma. While the function of S-100 protein is not known, a relationship between humoral factors related to pregnancy and expression of S-100 protein gene is suggested by the results of this study.

Comparison of Cytobrush and cervicovaginal lavage sampling methods for the detection of genital human papillomavirus.

The development of an accurate method for the detection and typing of genital human papillomavirus is of substantial clinical importance. This virus has been implicated as an etiologic agent in the development of cervical neoplasia. To detect human papillomavirus infection with maximum sensitivity, cells must be collected and assayed for human papillomavirus deoxyribonucleic acid. We compared two noninvasive methods of sampling exfoliated cervical cells--cervicovaginal lavage and scrape-Cytobrush. Seventy-four patients newly referred to the colposcopy clinic were divided randomly for cell sampling by either cervicovaginal lavage followed by scrape-Cytobrush or, conversely, scrape-Cytobrush followed by cervicovaginal lavage. Restriction analysis and Southern blot hybridization were used to test all the samples thus obtained for human papillomavirus. Overall, test results from 42 patients (56.8%) were positive for human papillomavirus deoxyribonucleic acid. Twenty-six (31.1%) tested positive for human papillomavirus by both sampling methods, and 32 (43.2%) tested negative for human papillomavirus by both methods. One (1.4%) tested positive with scrape-Cytobrush sampling but negative with cervicovaginal lavage, while 15 (20.3%) tested negative with scrape-Cytobrush but positive with cervicovaginal lavage (p less than 0.001, McNemar's test). These data, combined with previous work from our group, suggest that, of the available methods, cervicovaginal lavage, coupled with human papillomavirus deoxyribonucleic acid hybridization, is the most sensitive noninvasive method for harvesting cells for molecular identification of human papillomavirus in the female lower genital tract.

Changes in the connective tissue of corpus and cervix uteri during ripening and labour in term pregnancy.

The composition of the connective tissue of human cervix and corpus uteri was studied in tissue specimens from seven nonpregnant women and 14 pregnant women, delivered at term by section, to examine spontaneous cervical ripening and labour-induced changes in both the uterine and the cervical connective tissue. The main finding in both the cervix and the corpus was a large (40-60%) decrease of the collagen concentration. The collagen extractability, obtained by pepsin digestion, was increased twofold, suggesting a change of the organization of the collagen fibrils. This reorganization process could also be demonstrated by a large increase of the collagenolytic activity demonstrated with an artificial DNP-peptide substrate. The concentrations of sulphated glycosaminoglycans was lower in pregnant women than in non-pregnant women. The results show that both the cervix and the corpus uteri contain substantial amounts of connective tissue components (collagen, sulphated glycosaminoglycans and hyaluronic acid) and that during ripening, reconstruction of the connective tissue components occurs in both sites. This indicates that the cervical state reflects that of the myometrium.

Analysis of a 24-kilodalton (KD) protein in the human uterine cervix during abnormal growth.

The authors have previously studied the presence and distribution of a 24-kilodalton (KD) estrogen-regulated protein in the human normal cervix (Am J Obstet Gynecol 1986; 155:1090-1096). This protein has recently been identified as a heat-shock protein, and in order to continue its study the authors have now examined its expression in preneoplastic to neoplastic cervical samples. The study involved 53 patients, the presence of 24-KD protein together with keratin and carcinoembryonic antigen (CEA) was investigated by immunohistochemical analysis. Cytosol samples from 15 patients with squamous cervical carcinomas were also studied by the Western blot technique, and the presence of estrogen receptors was analyzed biochemically. The 24-KD protein was observed in cervical intraepithelial neoplasias (CIN), but it was not useful to identify the different degrees of CIN examined. The 24-KD protein, keratin, and CEA were predominantly expressed in well and moderately differentiated squamous carcinomas in the more differentiated areas, and the protein was also found in cervical adenocarcinomas. The presence of 24-KD protein did not correlate with that of estrogen receptors in squamous cervical carcinomas. The Western blot and the immunohistochemical studies revealed that the antibody to 24-KD protein does not cross-react with epitopes of CEA and keratins.

Is it safe to prescribe hormonal contraception and replacement therapy to patients with premalignant and malignant uterine cervices?

The levels of estrogen and progesterone receptors in normal and abnormal uterine cervices were determined. The study group consisted of 14 patients with cervical intraepithelial neoplasia (CIN III) and 7 patients with invasive carcinoma of the cervix (stage IB-IIA). The control group included 23 patients who underwent total abdominal hysterectomy for menorrhagia, leiomyoma, etc. The concentration of total estrogen receptors in premalignant and malignant cervices did not differ from the patients with benign conditions of the cervix. The concentration of progesterone receptors was significantly higher in the nonaffected cervices than in the patients with preinvasive and invasive carcinoma of the cervix (P less than 0.05). We have shown that estrogen receptor concentrations do not differ between women with normal and abnormal uterine cervices. Therefore, we feel that the contraceptive pill is not contraindicated in women who have been treated for CIN III. We also maintain that hormone replacement therapy should be given, when indicated, to women who have been castrated following surgery and/or radiotherapy for invasive carcinoma of the cervix.

Growth and characterization of epithelial cells from normal human uterine ectocervix and endocervix.

The human uterine cervix consists of an endocervical canal lined with a single layer of columnar mucus-secreting cells and an outer ectocervix covered by a stratified squamous epithelium. We report here the culture of human endocervical epithelial cells (HEnE) and human ectocervical epithelial cells (HEcE) in serum-free medium (KGM). Both HEnE and HEcE cultures were composed of keratinocytelike cells which formed desmosomal contacts and stratified in the presence of high concentrations of calcium ions. Cells with a pleomorphic epithelial morphology were observed in HEnE cultures, but not in HEcE cultures. Keratin 18, which is characteristic of endocervix in vivo, was detected by indirect immunofluorescent staining in all HEnE cells but was never detected in cultured HEcE. HEcE expressed keratin 13 which is characteristic of ectocervix in vivo. Although keratin 13 was never detected in primary HEnE cultures, it was expressed in passaged HEnE cultures grown in medium with high concentrations of calcium and in late passage HEnE cultures. HEnE underwent an average of 15.1 population doublings during serial culture. Mean colony-forming efficiency during Passages 2 to 3 was 14.7% and mean population doubling time was 17.8 h. HEcE cultures underwent significantly more population doublings (29.0) than HEnE cultures, whereas colony-forming efficiencies and doubling times were similar to those determined for HEnE. HEnE and HEcE cells may be useful in developing in vitro models of cervical squamous metaplasia and for exploring the interactions between target cell differentiation, carcinogens, and papillomaviruses in the development of cervical neoplasia.

Studies of lectin binding to the human cervix uteri: I. Normal cervix.

Lectins of Bauhinia purpurea (BPA), Canavalin ensiformis (Con A), Griffonia simplicifolia I (GS I), Griffonia simplicifolia II (GS II), Maclura pomifera (MPA), Arachis hypogaea (PNA), Glycine max (SBA), Ulex europaeus I (UEA I) and Triticum vulgaris (WGA) were used to evaluate cell surface carbohydrates in formalin-fixed paraffin-embedded tissue sections of normal human cervix uteri. Consistent patterns of staining of the squamous epithelium were obtained in all 30 cases with BPA, GS II, MPA, PNA, SBA and WGA. A variable distribution of lectin binding was seen in squamous epithelium with Con A, GS I and UEA I. The patterns of GS I and GS II binding reflected squamous epithelial maturation. Columnar epithelium did not stain with GS II, stained variably with Con A, and stained consistently with the remaining seven lectins in all cases. No association between lectin binding and blood group or phase of the menstrual cycle was found. These findings may be used as a baseline for evaluation of lectin binding in both preinvasive and invasive lesions of the cervix uteri.