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Objective : To investigate whether high-risk HPV infection associated with cervical intraepithelial neoplasia (CIN) was successfully eliminated after electrosurgical conization by large-loop excision of the transformation zone (LLETZ). Study Design : 142 women, who were admitted for conization of CIN 1–3 were recruited into a prospective follow-up study of HPV infection, including cervical sampling for HPV DNA before, and then 3, 6 and 12 months after surgery. We examined whether there were any differences in the rate of HPV DNA positivity after LLETZ between specific risk groups, such as patients with primary (i.e. before surgical treatment) high-risk HPV infection, CIN of different grades, and positive margins. Results : We did not detect statistically significant differences between specific risk groups. According to the assay used (hybrid capture II) at the last follow-up visit 94% of primarily infected patients were completely free from infection with high-risk HPV types, while 6% had persisting HPV infection. Conclusions : With a detection limit of 5000 genomes/ml HPV DNA the hybrid capture II results revealed, that after electrosurgical removal of CIN in 94% of patients testing positive for high-risk HPV DNA prior to surgery were negative 12 months post-surgery.

Objectives. The aim of this study was to investigate the impact of downregulation of KAI1 metastasis suppressor protein in epithelial ovarian cancer. In addition, correlation of KAI1 and p53 immunostaining was investigated. Methods. Expression of KAI1 and p53 was immunohistochemically determined in 107 specimens of epithelial ovarian cancer stages I–IV. Survival of patients was investigated using uni- and multivariate analysis. Results. Strong KAI1 expression was observed in 17.8% of cases, moderate in 27.1%, weak in 21.5%, and complete loss of KAI1 expression in 33.6%. Overexpression of p53 protein was observed in 45.8%. There was correlation of KAI1 expression neither with p53 expression nor with various clinical and histopathological parameters. Serous ovarian cancers showed significantly decreased staining intensity of KAI when compared to other histological types ( P = 0.007). Univariate and multivariate analysis revealed that patients with strong or moderate expression of KAI1 had a significantly longer overall ( P = 0.0013) and disease-free survival ( P = 0.0048) when compared to those with low or absent expression. Conclusion. KAI1 downregulation is an independent prognostic factor in epithelial ovarian cancer, indicating dismal prognosis. Our study did not reveal a correlation between p53 status and KAI1 expression, suggesting that p53-independent mechanisms might be involved in the downregulation of KAI1.

Detection and typing of human papillomavirus (HPV) infection may have a major impact in cervical-screening and follow-up. In this study various commercially available techniques for the detection of HPV were evaluated. HPV-status was determined in 86 samples of cervical cancer by PCR and direct sequencing, catalyzed signal amplified colorimetric DNA in situ hybridization (CSAC- ISH) (GenPoint system, DAKO), immunohistochemistry (IHC) and in 12 selected cases also by conventional, non-amplified ISH. Twenty-one samples of cervical intraepithelial neoplasias grade III (CIN III) were investigated by CSAC-ISH, conventional ISH and by IHC, in corresponding PAP smears HPV-detection and typing was performed by CSAC-ISH and Hybrid Capture test II (HC). In additional 20 PAP smears HPV typing was performed using HC and a novel immunocytochemical system for HPV detection and-typing. CSAC-ISH showed good correlation with PCR analysis in cervical cancers: In 87% of PCR positive cases, HPV infection was also detected by CSAC- ISH (66/76). HPV 16 was detected in 75% of PCR-positive cases (44/59), HPV 18 in 71% of PCR positive cases (5/7). CSAC-ISH detected HPV 31 in only 29% of PCR positive cases (2/7), and HPV 33 in 64% of PCR-positive cases (23/36). Nevertheless, CSAC-ISH- false negative cases for HPV 31 or 33 were nearly always combined infections with other HPV types, which were detectable by CSAC-ISH in most cases. CSAC-ISH revealed HPV infection in 20 of 21 HC-positive cervical smears, while in corresponding biopsies (CIN III) CSAC-ISH detected 100% of HPV infections. Conventional, non-amplified ISH showed significantly lower sensitivity compared with CSAC-ISH, and immunocyto- and -histochemistry were of very low sensitivity for detection of HPV. CSAC-ISH is an easy-to-handle method for detection and typing of cervical HPV infection, and shows sufficient sensitivity for clinical practice.

Downregulation of KAI1 metastasis suppressor protein is associated with dismal prognosis in a variety of cancers. Mutation of p53 was suggested to be involved in KAI1-downregulation. In cervical cancer, p53 is inactivated by human papillomavirus (HPV) oncoprotein E6 with the grade of inactivation depending on the HPV type. KAI1-expression was immunohistochemically determined in 67 specimens of cervical cancer, HPV-typing was performed using polymerase chain reaction (PCR), cloning, and sequencing. KAI1-downregulation was found in 68.1% of patients, HPV-infection in 91%. There was no association of KAI1-downregulation and infection with a particular HPV type. KAI1-downregulation in cervical cancer seems independent of HPV-E6 induced p53 inactivation.

Objective: Extrauterine leiomyomas are rare events. These tumors may be easily misdiagnosed as ovarian tumors at the clinical investigation. We present the first case of an otherwise healthy postmenopausal woman, hysterectomized 20 years ago, who developed a preperitoneal lipoleimoyoma in the 30-year-old scar of a Pfannenstiel incision. The patient received continuous hormone replacement therapy (HRT) for 5 years with 1.25 mg conjugated estrogen and 5 mg medrogeston per day. Methods: In sections of the tumor, immunohistochemical reactions with antibodies against actin, desmin, vimentin, estrogen and progesterone receptors and factor VIII related antigen was performed. Results: Histologic findings revealed cellular fascicles of spindle-shaped smooth muscle cells in a whorled arrangement. Mitotic figures were absent. Central degenerative changes and focal edema were observed. Between muscle fascicles, a significant amount of fat cells (20% of tumor volume) was visible. Leiomyocytes showed immunohistochemicaly positive reactions with actin, desmin, vimentin, and steroid hormone receptors. Based on these findings, the tumor was diagnosed as lipoleiomyoma. Conclusions: Origin of the tumor of smooth muscle cells of vessels located in the abdominal wall and development under influence of oral steroids seems most probable. HRT appears to promote the development of extrauterine leiomyomas in postmenopausal women.

Objectives. Fractal geometry is a tool used to characterize irregularly shaped and complex figures. It can be used not only to generate biological structures (e.g., the human renal artery tree), but also to derive parameters such as the fractal dimension in order to quantify the shapes of structures. As such, it allows user-independent evaluation and does not rely on the experience level of the examiner. Methods. We applied a box-counting algorithm to determine the fractal dimension of atypical nuclei in dysplastic cervical epithelium. An automatic algorithm was used to determine the fractal dimension of nuclei in order to prevent errors from manual segmentation. Four groups of patients (CIN 1–3 and control) with 10 subjects each were examined. In total, the fractal dimensions of 1200 nuclei were calculated. Results. We found that the fractal dimensions of the nuclei increased as the degree of dysplasia increased. There were significant differences between control and atypical nuclei found by an analysis of variance. Atypical nuclei associated with CIN 1, CIN 2, and CIN 3 also differed significantly among these groups. Conclusion. We conclude that the fractal dimension is a valuable tool for detecting irregularities in atypical nuclei of the cervix uteri and thus allows objective nuclear grading.

Tumor angiogenesis has been found to be prognostically significant in many types of malignant tumors. We assessed tumor vascularity in 43 cases of histologically proven primary fallopian tube cancer, FIGO stage I-IV, using the highly specific endothelial cell marker CD34. Microvessel count was determined by counting CD34-positive cells at 200 x magnification. The 5-year disease-free survival probability was 43.8% (+/- 11.5%) in 24 patients whose tumors had a microvessel countor = 19 microvessels/field and 19.7% (+/- 9.5%) in the19 microvessels/field group (P = 0.046). Stage and microvessel count were statistically significant for disease-free survival in univariate analysis. Therefore, a larger sample size would be required to detect an independent and statistically significant prognostic effect of microvessel density in primary fallopian tube cancer in multivariate analysis.

The expression of specific CD44 splice variants in malignant tumors has been shown to be associated with metastasis and poor prognosis. In previous studies we have detected aberrant expression of the transmembranous cell adhesion molecule CD44 in cervical cancer. Therefore, we now focus on its role in intraepithelial dysplastic lesions of the cervix. Forty-five cervical intraepithelial neoplasias (CIN), grades I to III, were investigated immunohistochemically for three different splice variants of CD44 (v5, v6, and v7-8). A negative correlation of CD44v5 (Kendall correlation coefficient r = -0.25, P < 0.05) expression with the grade of CIN was found. In normal squamous epithelium of the cervix, the staining reaction of CD44v5 was confined to the basal and the parabasal cell layers. In dysplastic lesions a diffuse and weaker staining pattern of the entire epithelium was observed. Our data suggest a loss of CD44v5 epitope during dysplastic transformation. It seems that expression of CD44 splice variants is not a continuous process in the natural history of cervical cancer, and the pattern of CD44 splice variant expression is changed during carcinogenesis.

Vulvar intraepithelial neoplasia (VIN) has been reported to be a precursor of invasive vulvar cancer. Switching to the angiogenic phenotype is considered a key step in tumor growth. Microvessel density (MVD) and vascular endothelial growth factor (VEGF), a highly angiogenic peptide, are important parameters of tumor angiogenesis. Forty-three histologic slides with 38 VIN I-III lesions were immunohistochemically stained for factor VIII-related antigen (F8-RA) and 44 slides with 37 VIN I-III for VEGF, since F8-RA reliably highlights tumor microvessels. Determination of MVD and VEGF expression was done by counting microvessels and VEGF-positive cells at a magnification of 200x and 400x. The highest concentration of F8-RA-stained MVD and VEGF expression was found at a small subepithelial area at the border of the VIN lesion to the stroma underneath but concentrations were low in all specimens of normal epithelium. High VEGF expression was significantly correlated to high MVD. For both MVD and VEGF expression the differences between VIN I and VIN III and between VIN II and VIN III were statistically significant (P < 0.0001). VIN III lesions are the clinical relevant precursors of invasive cancer of the vulva, as outlined by intense expression of VEGF protein and a highly dense network of microvessels underlying the dysplastic epithelium.

Microvessel density (MVD) and expression of vascular permeability factor/vascular endothelial growth factor (VPF/VEGF), acting as a highly specific inducer of angiogenesis, were evaluated in tissue specimens of 25 patients with squamous cell cancer of the vulva. MVD was quantified by immunostaining for factor VIII-related antigen at one field of 0.25 mm2. VPF/VEGF expression was evaluated immunohistochemically using a monoclonal anti-VEGF antibody. FIGO stages I, II, and III were diagnosed in 12, 7, and 6 patients, respectively. MVD >20/field was found in 10 of 25 tumors and moderate or strong expression of VPF/VEGF in 10 of 25 tumors. High MVD was significantly more frequent in tumors with moderate or strong VPF/VEGF expression compared to tumors with no or weak VPF/VEGF expression (P = 0.01). Overall survival rates of patients with tumors of high MVD (P = 0.01) and strong VPF/VEGF expression (P < 0.01) were significantly poorer compared to those patients with low MVD or poor VPF/VEGF expression. Strong VPF/VEGF expression and high MVD are considered important parameters of tumor angiogenesis and therefore are related to poor survival probability in vulvar cancer patients.

The expression of specific cell adhesion molecule CD44 isoforms (splice variants) has been shown to be associated with poor prognosis in human malignancies, such as breast cancer. We used three different variant exon sequence-specific murine monoclonal antibodies to epitopes encoded by exons v5, v6 or v7-v8 of human variant CD44, to study the expression of CD44 splice variants by immunohistochemistry in human stage III cervical cancer. We investigated 40 pretreatment punch biopsies of cervical cancer FIGO stage III. CD44 splice variants CD44v5, CD44v6 and CD44v7-8 were detected by means of immunohistochemistry in 90%, 55% and 25%, respectively. CD44 epitopes encoded by exon v5 were not correlated with prognosis. Expression of CD44 splice variants containing epitopes encoded by exon v6 were correlated with significantly poorer prognosis (Mantel test, P = 0.008). Five-year survival rates with or without CD44v6 expression were 20% versus 71%, respectively. Expression of CD44v7-8 was also correlated with significantly poorer overall survival (Mantel test, P = 0.02). Expression of CD44 splice variants containing epitopes encoded by exons v7-v8 and especially exon v6 is associated with significantly poorer prognosis in stage III cervical cancer patients.

Ch. Dittrich*, P. Sevelda, H. Salzer, A. Obermair, P. Speiser, G. Breitenecker, M. Schemper, A. Kaider on behalf of the Austrian Ovarian Cancer Study Group Ludwig Boltzmann-Institute for Applied Cancer Research, 3rd Medical Department with Oncology, Kaiser Franz Josef-Spital, Kundratstrasse 3, 1100 Vienna, Austria Department of Obstetrics and Gynecology, Krankenhaus Lainz, 1130 Vienna, Austria Department of Gynecology and Obstetrics, Wilhelminenspital, 1160 Vienna, Austria Department of Gynecology and Obstetrics, Vienna University, 1090 Vienna, Austria Department of Pathology, Gynecopathologic Unit, Vienna University, 1090 Vienna, Austria Department of Medical Computer Sciences, Vienna University, 1090 Vienna, Austria

Sections of formalin-fixed and paraffin-embedded tissue specimens of 11 normal ovaries and tubes, 13 tubo-ovarian abscesses, 3 tubal carcinomas, and 115 ovarian tumors were investigated by immunohistochemistry. CA 125 and CA 19-9 were demonstrated with monoclonal antibodies, CEA with polyclonal antibodies. The tissue expression was visualized by the avidin-biotin method. In the germinal epithelium of all ovaries no tumor marker was confirmed. In 4 out of 11 tubes the epithelium was CA 125 positive, in 2 out of 11 cases CA 19-9 positive. Nine out of 13 tubo-ovarian abscesses were CA 125 and 5 out of 13 were CA 19-9 positive in their epithelium. Elevated serum levels of these markers might be due to expression via the epithelial cell of the inflamed tube. All normal and inflammatory adnexal tissues were CEA negative. In serous tumors and undifferentiated carcinomas, CA 125 was most frequently confirmed (85 and 70%, respectively). All mucinous tumors were CA 125 negative. The most frequently confirmed tumor marker was CA 19-9 (77%). In endometrioid tumors, CEA was most frequent (44%). In 42% of the borderline tumors and carcinomas only one marker was demonstrated, in 7% none. Here, immunohistochemistry may indicate the most adequate marker. Tumor marker expression was markedly heterogenous: tumor areas with strong, weak, and no reaction were adjacent. The tumor markers revealed no specificity for malignancy or disease.

Human ovarian follicles showed activity of alkaline phosphatase, acid phosphatase (AcP) leucine aminopeptidase, and lactate dehydrogenase (LDH) in the theca interna. The granulosa of nonovulatory tertiary follicles showed moderate activity, whereas that of preovulatory Graafian follicles showed strong activity of LDH and AcP. The activity of these enzymes in the follicular fluid was measured. In nonovulatory tertiary follicles, activity of 3beta-ol-steroid dehydrogenase (3beta-OHSD) was found only in the theca interna; their fluid contained 290 ng/ml of progesterone and 502 ng/ml of 17beta-estradiol (average). Preovulatory Graafian follicles showed activity of 3beta-OHSD in the theca as well as in the granulosa. The progesterone concentration of the fluid was 7037 ng/ml and the 17beta-estradiol concentration was 2800 ng/ml (average). More oocytes could be aspirated from ovaries of younger women than from those of older women. In both age groups one out of three oocytes was degenerated. Oocytes with preovulatory changes were found only in follicles with preovulatory changes in their walls. Degenerated oocytes were found in some nonovulatory follicles as well as in some follicles with preovulatory changes in their walls.

CA 19-9, CA 125 and CEA were demonstrated by immunohistochemistry in 58 tissue samples of normal mucosa, 21 samples of atypical hyperplasia and 74 samples of endometrial carcinoma. CA 19-9 was mainly detected in the mid phase of secretion (8/11). CA 125 in the mid (6/11) and in the late phase (8/9). As opposed to CEA, both tumor markers are secretion products of the normal endometrium and are not expressed in the endometrial glands during the proliferation phase. CA 125 expression does not correlate with the degrees of differentiation or malignancy. The percentage of CA 19-9 positive cases rises with increasing differentiation. In atypical hyperplasia, however, this percentage is as small as in undifferentiated carcinoma. 93% of the endometrial carcinomas were CA 19-9, 65% CA 125 and 58% CEA positive.