Gisela Dallenbach-Hellweg

Overexpression of p16INK4a as a specific marker for dysplastic and neoplastic epithelial cells of the cervix uteri

Cytological screening for cervical cancer or its precursors using Papanicolaous smear test (Pap test) has been highly efficient to reduce the morbidity and mortality of cervical cancer. However, evaluation of the Pap test relies on subjective diagnostic parameters and is affected by a high rate of false‐positive and false‐negative results. More objective diagnostic parameters to identify truly dysplastic or neoplastic cells in cervical smears as well as in cervical biopsy samples would therefore avoid insecurity for many patients and the high screening costs associated with repeated testing. Cervical dysplasia is induced by persistent infections through high‐risk types of human papillomaviruses (HPVs). Outgrowth of dysplastic lesions is triggered by increasing expression of two viral oncogenes, E6 and E7, which both interact with various cell cycle‐regulating proteins. Among these is the retinoblastoma gene product pRB, which is inactivated by E7. pRB inhibits transcription of the cyclin‐dependent kinase inhibitor gene p16INK4a. Increasing expression of the viral oncogenes in dysplastic cervical cells might thus be reflected by increased expression of p16INK4a. In line with this hypothesis, we observed marked overexpression of p16INK4a in all cervical intraepithelial neoplasm (CIN) I lesions (n = 47) except those associated with low‐risk HPV types (n = 7), all CIN II lesions (n = 32), all CIN III lesions (n = 60) and 58 of 60 invasive cervical cancers. In contrast, no detectable expression of p16INK4a was observed in normal cervical epithelium (n = 42), inflammatory lesions (n = 48) and low‐grade cervical lesions (CIN I) associated with low‐risk HPV types (n = 7). Dysplastic cells could also be identified in cervical smears using a specific p16INK4a monoclonal antibody. These data demonstrate that p16INK4a is a specific biomarker to identify dysplastic cervical epithelia in sections of cervical biopsy samples or cervical smears.

Ovarian brenner tumors. I. Metaplastic, proliferating, and of low malignant potential

In several studies, attempts were made to establish criteria for distinguishing malignant Brenner tumors from proliferating and low malignant potential ones. Although these criteria can be applied to the majority of cases, there still exist tumors that present problems in classification. In applying the World Health Organization (WHO) criteria for Brenner tumors, the most important feature for distinguishing the intermediate forms from the malignant ones is the presence of stromal invasion in the latter. This feature has generally been considered difficult to employ because of the fundamental fibroepithelial nature of Brenner tumors, the stroma being derived from the ovarian stroma. A logical and relatively easily applicable classification of Brenner tumors is presented in this report. Although more complex than the WHO classification, it includes newly recognized variants of the Brenner tumor and avoids using the same terminology to describe different types and degrees of epithelial abnormalities. Fourteen unusual Brenner tumors were studied, intermediate between typical benign and frankly malignant ones, and were classified into 3 categories representing progressive epithelial abnormalities. These include metaplastic, proliferating, and tumors of low malignant potential. In none of these does stromal invasion occur. Each of these categories corresponds to a particular urothelial abnormality or neoplasm. Through this classification, a better understanding of the morphology and biologic behavior of unusual types of Brenner tumors can be expected.

Intermediate filaments of normal and neoplastic tissues of the female genital tract with emphasis on problems of differential tumor diagnosis

Cytokeratins of normal epithelia and of some neoplasms of the female genital tract were studied by immunofluorescence microscopy of frozen sections and by two-dimensional gel electrophoresis of cytoskeletal proteins from microdissected tissues. All normal epithelia were stained with the monoclonal cytokeratin antibody KG 8.13 whereas certain monoclonal antibodies stained only simple epithelia. As revealed by gel electrophoresis the normal epithelia of the ovarian surface, oviduct, endometrium and endocervix contained cytokeratin polypeptides Nos. 7, 8, 18 and 19. In contrast, stratified exocervical epithelium showed a much more complex pattern (polypeptides No. 1, 2, 4, 5, 6, 11, 13, 14, 15, 16, 17 and 19). A similar pattern was found in the vagina. All epithelial neoplasms studied, regardless of the degree of histologic differentiation, were stained with antibody KG 8.13 as well as with conventionally obtained guinea pig antibodies to bovine muzzle prekeratins. The ovarian, endometrial and endocervical epithelial tumors maintained the pattern of their cells of origin, i.e. they expressed only cytokeratins Nos. 7, 8, 18 and 19. In one type of endocervical adenocarcinoma an additional cytokeratin polypeptide (No. 17) was detected. In contrast, the epithelial tumors of the lower genital tract showed a more complex pattern which also showed some differences with respect to that described for the corresponding normal tissue. Thus, in non-keratinizing squamous cell cervical carcinomas, cytokeratins Nos. 5, 6, 7, 8, 13, 14, 15, 16, 17, 18 and 19 were present, whereas the keratinizing cervical cancers showed polypeptides Nos. 5, 6, 13, 14, 16, 17 and 19.(ABSTRACT TRUNCATED AT 250 WORDS)

Immunocytochemical study of an endometrial diffuse clear cell stromal sarcoma and other endometrial stromal sarcomas

Intermediate filament composition was studied in the following endometrial stromal tumors: low‐grade stromal sarcoma (endolymphatic stromal myosis), high‐grade stromal sarcoma with an associated adenocarcinoma (collision tumor), diffuse clear cell stromal sarcoma and a mesodermal mixed tumor (carcinosarcoma). The tumor cells of the stromal tumors as well as the mesenchymal elements of the mixed mesodermal tumor were decorated exclusively with antibodies to vimentin. Desmin was not demonstrated in these tumor cells. A biochemical study of the cytoskeletal filaments present in the low‐grade stromal sarcoma revealed, in addition to vimentin, β and γ actin as seen in normal endometrial stroma. Cytokeratins were only identified in epithelial components which were present in some of these tumors. Intermediate filament typing in these endometrial neoplasms contributes to the elucidation of histogenetic problems, may delineate mesenchymal from epithelial elements, may separate muscle from stromal lesions and in one instance helped to define a hitherto unreported diffuse clear cell stromal sarcoma.

The Normal Histology of the Endometrium

The endometrium of the corpus is composed of two layers: the basalis (the layer from which the endometrium regenerates after menstrual shedding) and the overlying functionalis. In the second half of the menstrual cycle, the functionalis may be differentiated into the superficial compacta and the underlying spongiosa, which extends to the basalis. During the menstrual cycle the endometrium varies from 1 mm (postmenstrual) to about 8 mm at the end of the 3rd week. Every layer consists of two major structures: the epithelial component, either as glands or as superficial epithelium, and the mesenchymal component of stromal cells. Both cellular components are pluripotential and can undergo various metaplastic changes.

Molecular analysis of Endometrial hyperplasia in HNPCC-suspicious patients may predict progression to Endometrial carcinoma

Women predisposed to hereditary nonpolyposis colorectal cancer are at high risk of developing endometrial carcinoma at a young age. Hereditary nonpolyposis colorectal cancer–associated endometrial carcinomas are of the endometrioid type, usually arise from complex atypical hyperplasia, and often show microsatellite instability. To identify occult hereditary nonpolyposis colorectal cancer individuals among endometrial carcinoma patients, we examined complex atypical hyperplasias and endometrial carcinomas of 60 women ≤50 years of age (mean age: 35.7 years) using microsatellite instability, immunohistochemistry, and DNA sequence analysis. Three patient groups were recruited: group 1, patients with complex atypical hyperplasia exclusively (n = 27); group 2, patients with complex atypical hyperplasia and synchronous or metachronous endometrial carcinoma (n = 15); group 3, patients with endometrial carcinoma only (n = 18). Overall, 13 of 33 endometrial carcinomas (39%) displayed high-level microsatellite instability. None of the complex atypical hyperplasias in group 1 had high-level microsatellite instability or loss of hMLH1/hMSH2 protein expression. In group 2 patients, 33% of complex atypical hyperplasias and 53% of endometrial carcinomas had high-level microsatellite instability. Loss of hMSH2 protein expression was found in six endometrial carcinoma patients, five of them with verified hMSH2 germline mutations, including four patients with high-level microsatellite instability in complex atypical hyperplasia. Among group 3 patients, 28% of endometrial carcinomas displayed high-level microsatellite instability; three of those five endometrial carcinomas were from patients with multiple extrauterine hereditary nonpolyposis colorectal cancer–associated tumors. We conclude that young women (≤50 years of age) with concurrent complex atypical hyperplasia and multiple hereditary nonpolyposis colorectal cancer–associated carcinomas are at risk of developing high-level microsatellite instability endometrial carcinoma. Combined microsatellite instability and immunohistochemistry analysis allows the identification of a high proportion of hereditary nonpolyposis colorectal cancer patients among young women with endometrial carcinoma and complex atypical hyperplasia. All complex atypical hyperplasias with high-level microsatellite instability progressed to endometrial carcinoma. Only one third of the complex atypical hyperplasias with microsatellite stability progressed to high-level microsatellite instability endometrial carcinoma, while seven complex atypical hyperplasias progressed to microsatellite stability endometrial carcinoma. Microsatellite analysis of complex atypical hyperplasia in young patients may therefore be a useful prognostic marker for predicting possible progression to high-level microsatellite instability endometrial carcinomas.

The Histopathology of the Endometrium

Almost all hormonal dysregulations (that is, endocrinopathies) and organic diseases of the endometrium produce atypical uterine bleeding. Clinically, the cause of the bleeding often remains unclear. Therefore, the attending gynecologist will Table 5 Causes of atypical uterine bleeding A. Systemic diseases: Cardiac and circulatory failure Hypertension Blood dyscrasia with thrombocytopenia Hemophilia, avitaminosis, intoxications, infectious diseases B. Functional disturbances : Dysfunction of the ovaries, pituitary, diencephalon, thyroid, or adrenal glands Psychogenic disturbances (cerebral) Death of extrauterine pregnancy Hormone-producing ovarian tumors Exogenous administration of hormones C. Local anatomic disturbances : Endometrium : Endometritis Abortion or retained products of conception Foreign bodies (talcum powder, intrauterine device) Polyps Neoplasms Myometrium : Vascular anomalies (aneurysms) Myometritis Fibromyoma-submucous, intramural, subserous Adenomyosis Neoplasms Portio and cervix: Cervicitis Polyps Glandular-papillary erosion Neoplasms Structural changes of the vagina, vulva, parametrium Anomalous positions of the uterus with circulatory disturbances (hemostasis) Usually can be diagnosed by histological study of curettings. Can be diagnosed only at times. attempt to establish a diagnosis in all his patients with unexplained bleeding by submitting curettings for histological study. Most causes of atypical uterine bleeding can be revealed histologically. The percentage of patients whose endometrial curettings fail to disclose pathological changes is small, varying from 3.6 per cent to 18 per cent, depending upon the investigator who makes the examination (Lau and Stoll, 1963; also for further literature). The cause of the bleeding in that small percentage of women is either morphologically extra-uterine or functionally extra-ovarian (see Table 5).

Screening for Cervical Cancer Precursors to Prevent Invasive Disease

Diagnostic or Therapeutic Procedures . . . . . . . . . . . . . . . . . . . . . . 2 Colposcopically Directed (Punch) Biopsy . . . . . . . . . . . . . . . . . . . 2 Cold Knife Conization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 Loop Electrosurgical Excision Procedure . . . . . . . . . . . . . . . . . . . 4 Endocervical Curettage . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4 Simple Hysterectomy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 Preparation of the Cervical Specimen . . . . . . . . . . . . . . . . . . . . . . 5

Characteristics of Fresh Cells in Phase-Contrast Microscopy

These are rounded cells with a distinct cytoplasmic membrane. The large, spherical nucleus usually is central and optically homogeneous. It contains one or two rounded nucleoli which appear dark because of their density. Delicate cytoplasmic granulations, like the occasional perinuclear vacuoles which either lie against the nucleus or surround it, apparently represent structures formed by metabolic changes. Through artifact (pressure on the coverslip, drying) cytoplasm may exude from the cells.

Gynecological Applications of Cytology

The gynecologist interested in morphology can extend the range of his or her routine diagnosis by phase-contrast microscopy of a fresh vaginal cell smear. Findings should be compared with anamnestic data and the results immediately discussed with the patient. Frequently, treatment needed can be initiated directly.