Gene expression in epithelial ovarian cancer: a study of intratumor heterogeneity.

The aim of this study was to investigate the intratumor heterogeneity of gene expression profiles in epithelial ovarian cancer (EOC). This was done to evaluate whether sampling of a single macrodissected tissue sample from each EOC case would bias the data and result in, eg, prognostic studies based on gene expression microarray experiments. From nine EOCs removed at Odense University Hospital, Denmark, three tumor samples of 200-300 mg each were taken with greatest possible mutual distance. The samples were immediately flash frozen. A parallel section was taken for histopathologic comparison. RNA was extracted from the tissue samples. Five micrograms of each RNA sample was used for labeling. The fragmented biotin-labeled complementary RNA was hybridized to Affymetrix GeneChip Human Genome U133 plus 2.0 arrays, and scanning was performed on the GeneArray scanner 3000 (Affymetrix, Santa Clara, CA). Data were evaluated using hierarchical clustering and intraclass correlation coefficient (ICC) from reliability analysis. All evaluation methods revealed low intratumor heterogeneity. Intratumor ICCs ranged from 0.888 to 0.978. In contrast, "between-tumor" ICC was 0.549 indicating much lower intra- than intertumor heterogeneity. Due to a low degree of intratumor variation, we conclude that it is sufficiently accurate in a clinical setup to use single, macrodissected tumor samples in the evaluation of gene expression in EOCs.

Prognostic significance of p53, Her-2, and EGFR overexpression in borderline and epithelial ovarian cancer.

The objective of the study was to evaluate the prognostic effect of p53, Her-2, and EGFR in borderline and epithelial ovarian cancer. Tumor tissue from 85 patients with borderline and 783 patients with epithelial ovarian cancer stage I-IV were analyzed immunohistochemically for p53 positivity and over-expression of Her-2 and EGFR. In the ovarian cancer (OC) group 415 patients (53%) had p53-positive tumors, 272 (35%) had tumors with Her-2 over-expression, and 483 (62%) had over-expression of EGFR. In the OC group the classical prognostic factors (older age, higher FIGO stage, and poorer differentiated stage) had significant prognostic value in both uni- and multivariate analyses. Multivariate analyses in the OC group proved p53 positivity to increase mortality significantly depending on the grade of the tumor. Her-2 likewise increased the risk of mortality significantly in this group depending on the grade of the tumor. EGFR on the other hand did not have any additional prognostic effect in the OC group after adjustment for the classical prognostic and molecular factors was made. In the borderline group Her-2 and EGFR over-expression in combination, adjusted for age and p53, significantly improved the prognosis.

Does plasminogen activator inhibitor-1 (PAI-1) control trophoblast invasion? A study of fetal and maternal tissue in intrauterine, tubal and molar pregnancies.

Urokinase plasminogen activator, its receptor and the inhibitor PAI-1 are believed to control proteolysis and remodelling of maternal tissue during trophoblast invasion. This system appears to be strictly regulated in normal intrauterine pregnancies whereas tubal and molar pregnancies seem to be characterized by an uncontrolled excessive placental invasion. This study evaluates subcellular PAI-1 by immunohistochemistry in the villous placenta, in the basal plate and placental bed, and in the decidual compartments of normal, tubal and molar pregnancies. PAI-1 was present in villous syncytiotrophoblasts and co-localized focally with fibrin-type fibrinoid on the surface of the chorionic villi. Basal plate and placental bed extravillous interstitial trophoblasts, as well as vascular trophoblasts, were also PAI-1 positive. In the decidua parietalis, PAI-1 was observed in the cytoplasm of the non-invaded decidual cells. In the decidua basalis comprising the basal plate, PAI-1 was seen to be membrane-associated or confined to the extracellular matrix (ECM) facing the invasive front of anchoring villi. The ECM of decidua capsularis and chorion laeve displayed the most pronounced PAI-1 expression towards the maternal interface. In contrast, the majority of placental bed decidual cells adjacent to the interstitial and vascular trophoblasts were PAI-1 negative. Only a few stromal cells distant from the implantation site were PAI-1 positive in the tubal pregnancies and decidualization was not present. Likewise, excessive decidual necrosis and fibrinoid deposition devoid of PAI-1 was a common finding in complete molar pregnancies. These results suggest that PAI-1 defines specific extravillous invasive trophoblasts within the maternal decidua. Moreover, maternal cellular lack of PAI-1 in tubal pregnancies and excessive decidual necrosis in molar pregnancies indicate an uncontrolled placental invasion. The present data indicate that trophoblast invasion is primarily regulated by signals from decidual cells.

Localization of E-cadherin in villous, extravillous and vascular trophoblasts during intrauterine, ectopic and molar pregnancy.

Previous reports have described down-regulation of E-cadherin in trophoblasts differentiating to an invasive phenotype. This study shows the localization of E-cadherin in a prospective design with stereological sampling of fetal and maternal first, second and third trimester tissue. E-cadherin was observed in villous cytotrophoblasts, and in non-proliferating, intermediate trophoblasts (IT) within cell columns and islands in intrauterine, ectopic and partial molar placentas. Highly proliferating IT with cytological atypia in complete molar placentas were also E-cadherin-positive. E-cadherin was present in trophoblasts throughout the anchoring cell columns. Trophoblasts undergoing epithelial mesenchymal transformation (EMT) detaching from the distal cell columns and deeper located single extravillous interstitial trophoblasts (EVT) showed E-cadherin-negative breaches in the cell membrane. Prior to the late second trimester, the relative number of E-cadherin-positive single EMT and EVT differed from the total number of cytokeratin-positive trophoblasts. Intraluminal, endovascular and perivascular trophoblasts adjacent to the maternal vessels were also E-cadherin-positive, but a highly varying pattern was observed at different ages of gestation. Our results indicate a temporary shift in E-cadherin expression in extravillous trophoblasts possessing a migrating and invasive potential. Functional E-cadherin may be restored as trophoblasts aggregate in the decidua and the vessel wall after completion of migration.

Mucinous cystadenocarcinoma in combination with hemangiosarcoma in the ovary.

The ovary is the sixth most frequent site of cancer in women in Denmark with an incidence of approximately 600 cases per year. Carcinomas predominate whereas sarcomas are rare. We describe a case of the combination mucinous cystadenocarcinoma and hemangiosarcoma in a 37-year old woman, who had a right-sited oophorectomy because of a cyst. Clinically there was no suspicion of malignancy. The macro- and microscopic findings are described as well as the immunohistochemical stainings performed to confirm the diagnosis. The case shows the importance of careful sampling at the macroscopic examination, especially from areas with a striking appearance.

Efficacy of Ki-67 antigen staining in Papanicolaou (Pap) smears in post-menopausal women with atypia--an audit.

In cervical smears from post-menopausal women with mucosal atrophy it can be difficult to distinguish atrophic epithelial cell groups from neoplastic cell groups on cytomorphological criteria only. The consequence of post-menopausal atypia is that the woman is referred for a repeat smear after local oestrogen treatment or for colposcopy. We investigated whether immunocytochemical expression of Ki-67 (MIB-1) on the primary Papanicolaou-stained smear could be of any diagnostic help. Our data showed that negative Ki-67 expression is a very reliable indicator of a normal atrophic cell pattern, and by using this method on the original smear we were able to reduce the false-positive cytologic diagnoses by 86%.

Localization and significance of urokinase plasminogen activator and its receptor in placental tissue from intrauterine, ectopic and molar pregnancies.

Urokinase plasminogen activator receptor (uPAR) is a membrane-anchored protein with urokinase plasminogen activator (uPA) as the ligand. This complex induces proteolysis and remodelling of maternal decidua during placental implantation. The presence of uPAR on trophoblasts is supposed to promote adhesion, migration and invasion. In cancer tissue, high levels of uPAR are correlated with a poor prognosis. This immunohistochemical study shows the localization of uPA and uPAR in a prospective design with stereological sampling of fetal and maternal tissue from normal, ectopic and hydatidiform molar (HM) pregnancies. Cytokeratin and Ki67 were used as markers for trophoblasts and proliferating cells. Membrane-bound uPAR was observed on villous non-proliferating intermediate trophoblasts (IT) within cell columns in intrauterine and ectopic pregnancies. The corresponding proliferating IT with cytological atypia sprouting from the chorionic villi in HM was uPAR-negative. uPA but not uPAR was observed in anchoring distal IT at the attachment-point to the basal plate. In the placental bed, extravillous interstitial trophoblasts were uPA-positive but uPAR-negative. The trophoblast giant cells were both uPA- and uPAR-negative. In relation to the maternal vessels, a focal distribution for uPA and uPAR was present in the endovascular and perivascular trophoblasts. The intraluminal trophoblasts overlying endothelial cells were uPAR-positive only. In maternal tissue from intrauterine and molar pregnancies, uPAR was seen in the decidual cells in a zone facing the anchoring villi and the fibrinoid lesions with embedded trophoblasts. In contrast, the stromal cells of the fallopian tube without a decidual reaction facing the implanted gestation were uPAR-negative. Non-invaded decidual, myometrial and muscular tissue of the pregnant uterus and fallopian tube was extensively positive for uPA whereas 'pseudodecidual' cells from the intrauterine evacuate in patients with an ectopic pregnancy only showed a focal and scanty reaction for uPA. When trophoblast invasion of the decidua was present, the decidual cells were uPA-negative. A semi-quantitative assessment of the receptor was estimated in villous IT within cell columns in normal and molar pregnancies but, in conclusion, quantitative evaluation of uPAR cannot be used to predict development of post-molar persistent trophoblastic disease (PTD).

[Follow-up of non-negative cervical cytological smears in the county of Funen]. / Opfølgning af ikkenegative cervixcytologiske prøver i Fyns Amt.

The outcome of screening for cervical cancer in the county of Funen was evaluated in two sequential periods (1.7.-31.12.1989 and 1.7.-31.12.1992), comprising 17,493 and 18,135 respectively. About 10.5% of the screened women had a define non-negative smear. From the first and the second period 80% and 85.1% of the non-negative smears respectively were followed up within six months. The follow-up of positive smears was 96% in both periods. Four point nine percent and 3.3% respectively of the non-negative smears were not followed up within 18 months. No women were actually lost in the screening system, unless they renounced further follow-up themselves. The follow-up was improved from the first to the second period, presumably as a result of a better general acquaintance with the screening procedures. The study indicates that reorganization of a screening programme requires both time and adjustment. Moreover, it is important that a successful screening programme frequently adjusts its procedures.

[Evaluation of PAPNET--a semiautomated system used in the screening against cervical cancer]. / Evaluering af PAPNET--et semiautomatisk screeningssystems anvendelse i screening mod livmoderhalskraeft.

The PAPNET-system is a current example of automated technological progress in the pathological laboratory field. As the first Department of Pathology in Denmark, we have tested the applicability of this semi-automatical screening system in screening against cervical cancer. 3000 prospectively selected cervical smears were entered into the project. 1500 of these were first prescreened by the use of PAPNET and the negative slides were then manually rescreened. The remaining 1500 slides consisted of manually screened smears diagnosed as negative or inadequate. They were subsequently rescreened by the use of PAPNET. We only found one false negative smear in each group. Compared with histological follow-up the diagnoses CIN 1-3 were histologically confirmed in both groups. The PAPNET-assisted screening of cervical smears is faster, more valid and less fatiguing than the conventional screening method. Nevertheless, our results show no diagnostic quality improvement by the use of PAPNET. This is probably due to a strict screening procedure and a limited work load of a maximum of about 40-50 slides per cytotechnologist a day in our laboratory.

Primitive neuroectodermal tumor (PNET) of the uterine cavity.

The occurrence of primitive neuroectodermal tumors located in the uterus is extremely rare. Eight cases have been described in the literature, and with the addition of this ninth case, we summarize treatment and outcome of PNET located in the uterine cavity.

[Screening against cervical cancer. Experiences, attitudes and knowledge of women in the county of Funen]. / Screening mod livmoderhalskraeft. Fynske kvinders erfaringer, holdninger og viden.

Questionnaires on women's attitudes and knowledge of cervical screening in the County of Funen were mailed to a sample of 1505 attenders aged 23-59 years, stratified on age and residence. A high proportion of attenders (80.4%) answered the questionnaire. Two-thirds of the women had been informed about the screening program before they received the invitation to participate. Generally the women were satisfied with the introduction to the screening program. However, the majority of the attenders were not satisfied with the way they received the result. They wanted the result no later than two weeks after the test was done, and they wanted the result from their GP. Independent of age and education the majority (about 90%) had a good general knowledge (knew that the smear is taken from the cervix; that early diagnosis is important; that cervix cancer can be treated). However the specific knowledge was lower and significantly associated with education (e.g. 43% versus 63% could identify the cervix on a drawing of the uterus among women with respectively low and high levels of school education). Furthermore, the majority were of the opinion that the test should be done more frequently than every three years as recommended by the National Health Board.

[Patients with early stages of endometrial cancer should be spared adjuvant radiotherapy. Danish Endometrial Cancer Group]. / Patienter med tidlige stadier af endometriecancer bør skånes for adjuverende strålebehandling.

In an attempt to create uniform nationwide guidelines for the management of all stages of endometrial carcinoma, and to limit the use of adjuvant radiation therapy in stage I disease to high-risk patients only, a protocol was developed by the Danish Endometrial Cancer group (DEMCA). From September 1986 through August 1988, 1214 women in Denmark with newly diagnosed carcinoma of the endometrium have been treated according to this protocol. This figure represents all endometrial carcinomas diagnosed in Denmark during this two-year period. The primary treatment was total abdominal hysterectomy and bilateral salpingo-oophorectomy, no preoperative radiation therapy was delivered. In 1039 cases no macroscopic residual tumour and/or microscopic tumor tissue in the resection margins was found following surgery. Based on surgery and histopathology, these patients were classified as: P-stage I low risk (n = 641), P-stage I high risk (n = 235), P-stage II (n = 105) and P-stage III, Group 1 (n = 58). No postoperative radiation therapy was given to P-I low risk cases. P-I high risk, P-II, and P-III (Group 1) cases received external radiation therapy. Recurrence rate at 68-92 months follow-up was 45/641 (7%) in P-I low risk, 36/235 (15%) in P-I high risk, 30/105 (29%) in P-II, and 27/58 (47%) in P-III (Group 1) cases. Fifteen of 17 vaginal recurrences in P-I low risk cases were salvaged (mean observation time 61 months). In this population-based investigation it has been shown that P-stage low-risk patients are adequately treated by total abdominal hysterectomy and bilateral salpingo-oophorectomy, and that no pre- or postoperative radiation therapy is necessary.

Immunohistochemistry (Ki-67 and p53) as a tool in determining malignancy in smooth muscle neoplasms (exemplified by a myxoid leiomyosarcoma of the uterus).

Smooth muscle tumours of the uterus at times represent a problem as it may be difficult to distinguish between benign and malignant tumours. The myxoid leiomyosarcoma (a rare type of neoplasm) reported here is an example of this. We present a case history and examine the suitability of Ki-67 and p53 as indicators of malignancy. The two antibodies were tested on seven leiomyomas, three atypical (borderline) leiomyomas, seven leiomyosarcomas and the myxoid leiomyosarcoma using microwave oven antigen retrieval. The leiomyomas had the lowest and the leiomyosarcomas the highest proliferation rate. The leiomyomas had no expression of p53, the atypical leiomyomas had a few scattered positive nuclei, and 5/7 of the leiomyosarcomas had overexpression of p53. The myxoid leiomyosarcoma had a positive reaction for p53 in clusters. The results suggest that Ki-67 and p53 might be useful as indicators of malignancy.

[Costs and effects of alternative screening programs against cervical cancer]. / Omkostninger og effekter af alternative screeningsprogrammer mod livmoderhalskraeft.

This paper presents a framework for comparison of screening programme designs, based on efficiency and cost effectiveness criteria. Design parameters such as choice of screening interval and which population segments to screen are varied simultaneously. The costs and effects for a range of existing and hypothetical screening programmes for cervical cancer are estimated, using a mathematical simulation model. On the basis of these estimations incremental costs per life year are calculated for a range of programmes. Efficiency and cost effectiveness criteria indicate that extending screening programmes for cervical cancer beyond screening women in the age group 25-59 years every four years may not be optimal.

[Atypical cervix uteri smears--consequences?]. / Cytologisk atypi i cervix uteri--konsekvens?

Among 37,992 cervical smears (725 (1.9%)) were found to be atypical. All atypial smears were referred for colposcopically guided biopsies and cervical abrasio within three months. This is an expensive and cumbersome procedure. In order to obtain a more differentiated follow-up procedure the atypical smears were divided into two groups without knowledge of the later histological diagnosis on biopsy: "Atypia, probably reactive" and "atypia, probably CIN (cervical intraepithelial neoplasia)". In the latter group the subsequent biopsies showed significantly more CIN than in the former (60.4% against 15.7%). We find it useful to divide the atypical smears into two groups in our daily routine work with a different follow-up procedure: "Atypia, probably reactive" to be followed by a repeat smear and "atypia, probably CIN" to be followed by colposcopically guided biopsies and cervical abrasio.

Ectopic pregnancy: histopathology and assessment of cell proliferation with and without methotrexate treatment.

OBJECTIVE: To evaluate tubal morphology, trophoblast proliferation, and inflammatory reaction in response to methotrexate (MTX) treatment of ectopic pregnancy (EP). DESIGN: Nonrandomized controlled study. SETTING: Academic hospital. PATIENTS: Archival specimens from 10 EP unsuccessfully treated with MTX and 10 cases primarily treated by surgery. INTERVENTIONS: Ki67/hCG and Ki67/human placental lactogen double immunohistochemical methods were used to examine trophoblastic spread, placentation, hormone production, decidualization, vascular invasion, hemorrhage, rupture, and proliferative index of the cytotrophoblast. B and T-lymphocyte responses were evaluated by CD3 and CD20. RESULTS: Trophoblastic spread and placentation were confined to the tubal mucosa after MTX treatment, whereas invasion of the muscularis and subserosa was common in the controls. The proliferative index was reduced (19 percent versus 93 percent), although a high proliferative index was found in two of three cases complicated by rupture. Polar proliferation of Ki67-positive cytotrophoblast toward the implantation site was abolished in MTX-treated cases. Decidual reaction was not observed. No correlation was observed between the above-mentioned findings and gestational age, level of beta-hCG, dose of MTX, or interval to surgery. CONCLUSION: Trophoblastic spread, differentiation, and invasion were compromised by MTX treatment. Methotrexate seems to decrease cytotrophoblast proliferation. Whether a missing decrease in proliferation index reflects treatment failure awaits a larger population-based study.

A cost-effectiveness analysis of cervical cancer screening: health policy implications.

This paper presents a framework for comparison of screening programme designs, based on efficiency and cost effectiveness criteria. The design parameters, such as choice of screening interval, which population segments to screen and expected participation rates in the selected population segments, are varied simultaneously. The costs and effects for a range of existing and hypothetical screening programmes against cervical cancer are estimated, using a mathematical simulation model. On the basis of these estimates average costs per life year and marginal costs per life year are calculated for a range of programmes. These calculations result in the definition of a range of inefficient programmes. Moreover, it is illustrated that the cost effectiveness of the efficient screening programmes decreases at an increasing rate as programmes are intensified either by way of shortening the screening interval or extending the target population segment to encompass the very young and/or the very old. The conclusion of this paper is that one should probably not extend screening programmes against cervical cancer beyond screening women in the age group 25-59 years every 4 years. In addition, increasing the participation rate of this group is a more cost effective way of increasing the number of life years gained, rather than extending the target group or decreasing the screening interval.

Near-diploid karyotypes with recurrent chromosome abnormalities characterize early-stage endometrial cancer.

Cytogenetic investigation was attempted on 15 endometrial tumors. Whenever possible, a combination of direct harvesting and short-term culture (with or without prior methotrexate synchronization) was used. The analysis was successful in 13 cases: 12 carcinomas of stage I and one atypical hyperplasia. Clonal abnormalities were found in 10 tumors, whereas the remaining three showed a normal karyotype. The modal chromosome number was near-diploid. The abnormal karyotypes contained relatively simple numerical or structural aberrations in all but one tumor, a serous papillary carcinoma with multiple complex changes as well as cytogenetic evidence of intratumor heterogeneity. Gain of 1q, trisomy for chromosomes 2, 7, 10 (this trisomy was shown by in situ hybridization to be present also in a large number of interphase cells), and 12, and loss of chromosome 22 were recurrent aberrations; these are also the cytogenetic anomalies that have been consistently associated with endometrial carcinomas in previous studies. The utilization of both direct harvesting and short-term culture in several cases increased the frequency with which abnormal karyotypes were found; sometimes aberrations were found by the first method but not by the other, and vice versa. Never were different clonal anomalies found by the two approaches in the same case. Synchronization of the cultures generally led to chromosome preparations with more mitoses and of better quality. Again, no different anomalies were found in synchronized and standard cultures from the same tumor.

Neural network processing of cervical smears can lead to a decrease in diagnostic variability and an increase in screening efficacy: a study of 63 false-negative smears.

A realistic approach for decreasing the number of erroneous diagnoses plaguing cervical cytology screening is to try to reduce the amount of nondiagnostic visual information. The neural network of PAPNET selects 128 cytological views from the routinely prepared smear which in digitized form can be displayed on a high-resolution videoscreen. From these 128 videotiles the abnormal ones can be selected by the diagnostician and brought together on the "summarizing videoscreen" containing 16 tiles. Thus, the diagnostic information can be further condensed. This facilitates the proper interpretation of the diagnostic cell material dispersed over the smear. A series of 63 false-negative smears were rescreened twice conventionally and twice using the PAPNET-assisted method. We found that, using PAPNET, the screening efficacy increased and the diagnostic variability decreased. The PAPNET in particular proved to be superior for smears containing few abnormal cells and cases of malignancies of the reserve cell lineage.