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Objective: To analyze the expression of Heparanase, an enzyme involved in cancer metastasis and angiogenesis, in ovarian and breast carcinoma cells in effusions., Methods: Heparanase protein expression was analyzed in malignant effusions from ovarian (=200) and breast (=41) carcinoma patients using immunocytochemistry. The levels of secreted heparanase were analyzed in 45 effusion supernatants using a newly established ELISA test. Heparanase expression levels were analyzed for clinical significance., Results: Heparanase was expressed at the cell membrane in 106/200 (53%) ovarian and 22/41 (54%) breast carcinomas. Cytoplasmic expression was found in 180/200 (90%) ovarian and 26/41 (63%) breast carcinomas. Reactive mesothelial cells showed frequent cytoplasmic, but not membrane expression. ELISA showed secreted heparanase in all 45 analyzed effusions. Higher levels were detected in peritoneal compared to pleural effusions (p=0.031). In univariate survival analysis of ovarian carcinoma patients with post-chemotherapy effusions, membrane expression in >5% of tumor cells correlated with shorter overall survival (OS, p=0.013). FIGO stage (p=0.03 for all patients, p=0.045 for those with post-chemotherapy specimens) and response to first-line chemotherapy (p<0.0001 for all patients, p=0.049 for those with post-chemotherapy specimens) were the clinical parameters related to OS. In Cox analysis of this subset of patients, heparanase expression (p=0.02) and response to chemotherapy (p=0.049) were independent predictors of poor OS. Heparanase expression did not correlate with survival in breast carcinoma., Conclusions: Our data show that heparanase is frequently expressed in metastatic gynecologic adenocarcinomas, and that it is secreted into the effusion fluid in body cavities. The correlation between heparanase expression and poor survival in ovarian carcinoma suggests a role for this molecule in ovarian cancer metastasis and supports its role as a marker of aggressive clinical behavior at disease recurrence.

Objective: To compare DNA-based and mRNA-based methods for detection of high-grade cervical neoplasia in Norway., Methods: HPV prevalence was analyzed in 383 women with positive index cytology, selected from gynecology clinics. All patients were investigated by a new PAP smear, histology, and two commercially available HPV tests: Hybrid Capture II (Digene, Gaithersburg, MD) and the Pre Tect HPV-Proofer (NorChip AS). Cases with positive DNA test and negative mRNA test and cases with high-grade histology and negative HPV tests were retested with PCR and sequencing. We regarded the infection as latent or transient if sequencing revealed an HPV type included in both assays., Results: High-risk HPV was detected in 99.7% of the histological confirmed high-grade lesions (CIN2+) (290/291). The DNA test was positive in 95% (275/291), and the mRNA test was positive in 77% (225/291) of the histological confirmed high-grade lesions. All invasive carcinomas were mRNA positive. The DNA test was significantly more often positive in benign and low-grade lesions, some of which were found to be false positive due to cross-contamination with unrelated types. High-grade histology was detected in 83% of women with normal cytology and positive mRNA test. Latent or transient infections were detected in 11 low-grade and 12 high-grade preinvasive lesions. Sequencing revealed high-risk HPV types included only in the DNA test in 35 high-grade preinvasive lesions, HPV 52 and 58 were the most prevalent HPV types., Conclusions: These HPV tests have the potential to improve the detection rate of high-grade cervical neoplasia, with some limitations. The mRNA test seems to be more appropriate for risk-evaluation. Larger scale, population based studies are necessary to evaluate the predictive values of HPV testing in Norway.

Objective: We recently showed that the levels of secreted human leukocyte antigen-G (HLA-G), a nonclassical MHC class I antigen, are significantly elevated in malignant effusions in ovarian carcinoma compared to benign ones. The objective of this study was to evaluate the expression and clinical role of HLA-G in effusions and corresponding solid tumors from patients diagnosed with advanced-stage ovarian carcinoma., Methods: Effusions (= 148), corresponding primary tumors (= 66), and metastatic lesions (= 122) were analyzed using immunohistochemistry with an anti-HLA-G monoclonal antibody., Results: HLA-G was detected in cancer cells in 49/148 (33%) effusions, 33/66 (50%) primary tumors, and 59/122 (48%) solid metastases. These differences did not reach statistical significance. Expression in effusions and solid metastases significantly correlated (P = 0.029). HLA-G expression in tumor cells was significantly lower in effusions obtained during or following chemotherapy (P = 0.038). The presence of HLA-G-positive tumor cells in effusions obtained prior to the institution of chemotherapy correlated with better overall survival (P = 0.042). HLA-G expression in primary tumors and solid metastases did not correlate with any of the clinicopathologic parameters studied., Conclusions: HLA-G is expressed in a significant number of ovarian carcinomas at all anatomic sites. The reduced expression of HLA-G in post-chemotherapy effusions and its correlation with improved survival may be related to preferential susceptibility of HLA-G-expressing cells at this site. Our findings suggest a new role for HLA-G as a prognostic indicator in advanced-stage ovarian cancer in effusions.

Objective: Carcinoma of the ovary is the most fatal malignancy of the female genital tract in western countries. The effectiveness of chemotherapy is limited by the acquisition of drug resistance. Several members of the kallikrein (KLK) family were recently shown to be expressed in ovarian cancer and implicated in disease prognosis. One of these is KLK4, whose increased mRNA expression was identified as a poor prognostic marker in ovarian cancer. The goal of this study was to investigate if KLK4 protein (hK4) is expressed in ovarian carcinoma lesions and if it is associated with paclitaxel resistance., Methods: Immunohistochemistry was used to assess hK4 expression. The 126 lesions were from a cohort of 46 patients with platinum-resistant tumors, which were treated with weekly paclitaxel for recurrent disease. The overall response rate to weekly paclitaxel was 52% (24 of 46) and for patients with tumors resistant to standard three weekly paclitaxel treatment 48% (16 of 33)., Results: Immunohistochemistry indicated that 85% (79 of 93) of lesions from paclitaxel-resistant patients expressed hK4, while only 61% (20 of 33) of lesions were positive for hK4 from paclitaxel sensitive patients. Statistical analysis showed that the intensity of hK4 staining was significantly associated with paclitaxel resistance (P = 0.005), whereas hK4 staining extent showed marginal significance (P = 0.05), but was significantly correlated with higher histological grade (P < 0.001)., Conclusion: These data show that hK4 is expressed in ovarian cancer and suggest that hK4 expression might be a predictive marker for paclitaxel resistance.

Objective: The objective was to study expression of alphav- and beta1-integrin subunits in effusions, primary tumors, and solid metastases of ovarian carcinoma patients, as well as to evaluate its potential association with previously studied metastasis-associated molecules and clinicopathologic parameters., Methods: Sections from 121 malignant effusions and 30 corresponding primary and metastatic lesions were evaluated for protein expression of the alphav- and beta1-integrin subunits using immunohistochemistry (IHC). A subset of effusions was additionally studied using immunoblotting (IB) and flow cytometry (FCM). mRNA in situ hybridization (ISH) was performed in 58 effusions and 30 biopsies., Results: Protein expression of alphav- and beta1-integrin subunits was detected in carcinoma cells in 116/121 (96%) and 113/121 (93%) effusions, respectively. alphav protein expression was limited to carcinoma cells. IB and FCM confirmed IHC results. mRNA for alphav- and beta1-integrin subunits was detected in carcinoma cells in 37/58 (64%) and 33/58 (57%) effusions, respectively. Both protein and mRNA expression were higher in peritoneal effusions, significantly for alphav mRNA (P = 0.042) and beta1 protein (P = 0.023). beta1 protein expression in effusions was more frequently detected in better-differentiated tumors (P = 0.006). alphav-integrin subunit expression correlated with that of the previously studied matrix metalloproteinase-9 (MMP-9) (P = 0.006) and the MMP inducer EMMPRIN (P = 0.001). Expression of beta1-integrin subunit showed an association with that of EMMPRIN (P = 0.029), basic fibroblast growth factor (P < 0.001), and the MMP inhibitor TIMP-2 (P = 0.025). In carcinoma cells of solid lesions, alphav protein was uniformly present, while beta1 expression was limited to 15/30 (50%) specimens. As in effusions, protein expression of alphav subunit was cancer-specific, while beta1 protein was detected also in stromal fibroblasts and endothelial cells., Conclusions: The alphav- and beta1-integrin subunits are frequently expressed in ovarian carcinoma cells in effusions, and the alphav-integrin subunit is a powerful diagnostic marker for carcinoma cells. The reduced expression of the beta1-integrin subunit in solid lesions may be attributed to the role of other subunits at these stages, such as the beta3 subunit as part of the alphavbeta3-vitronectin receptor. The high expression of integrin subunits with a role of binding mesothelium, invasion, and angiogenesis in carcinoma cells in both peritoneal and pleural effusions suggests that cells at both sites have metastatic potential.

Objective: The aim of this study was to investigate the expression of cell cycle proteins in ovarian carcinoma cells in serous effusions and respective solid tumors., Methods: Fifty-five malignant effusions and 38 tumors (20 primary, 18 metastatic) were immunohistochemically stained for cyclin A, p27(kip1), and Ki-67. Staining extent (0-100% cells) and intensity (0-3 scale) were scored. Cyclin A and p27(kip1) expression was additionally studied in 29 malignant effusions using immunoblotting. Immunohistochemistry results in effusions were evaluated for possible association with clinicopathologic parameters., Results: Nuclear immunoreactivity for all markers was detected on carcinoma cells in the majority of effusions using immunohistochemistry. Similarly, immunoblotting showed the presence of cyclin A and p27(kip1) in 29/29 and 25/29 specimens, respectively. Intense (3) immunoreactivity for Ki-67 was detected more often in peritoneal effusions, compared with those of pleural location (P = 0.036). Staining in primary and metastatic lesions was generally comparable to that of tumor cells in effusions. Staining for p27(kip1) was more diffuse in effusion specimens obtained prior to the institution of chemotherapy (P = 0.042). In an analysis of all effusions, an association was observed between the number of cells that were immunoreactive for Ki-67, cyclin A, and p27(kip1) (cyclin A-Ki-67: P = 0.008; p27(kip1)-Ki-67: P = 0.019; cyclin A-p27(kip1): P = 0.032). In survival analysis, the presence of more diffuse (P = 0.042) and intense (P = 0.019) staining for cyclin A correlated with prolonged overall survival., Conclusions: The expression of the studied cell cycle markers does not differ markedly between ovarian carcinoma cells in the pleural and peritoneal cavity, supporting our previous studies of several metastasis-associated molecules. The presence of cyclin-A-positive cell populations is associated with a more favorable disease outcome, possibly due to the targeting of proliferating cells by chemotherapeutic agents. However, the decline in the fraction of p27(kip1)-positive cells in posttreatment specimens may point to additional mechanisms involved in this selection., (Copyright 2001 Academic Press.)

Objective: The objective of this study was to evaluate the prognostic significance of clinical and histopathologic factors, including a new grading system focusing on the invasive tumor front., Method: A retrospective analysis of 125 surgically treated patients with squamous cell cervical carcinoma FIGO stage IB was conducted. For each tumor, the degree of keratinization, nuclear polymorphism, pattern of invasion, and degree of lymphoid infiltration at the invasive tumor front were graded and given scores between 1 and 4., Results: Clinical tumor size, depth of invasion, and grading of the invasive front had prognostic significance in multivariate analysis, while lymph vascular space involvement, lymph node status, and grade of differentiation did not. Based on clinical tumor size, depth of invasion, and grading of the invasive tumor front, patients could be separated into three groups: One group with minimal risk of recurrence (5-year disease-free survival (DFS) of 100%) consisting of 24% of the patients, an intermediate group with a fairly low risk of recurrence (5-year DFS of about 92%), and a high risk group with a 5-year DFS of 45%. This latter group contained 26% of the patients with 78% of all relapses occurring in the total group of patients. The invasive tumor front grading was reliably reproducible, with inter- and intraobserver agreement of 79 to 87% and kappa values of 0.47 to 0.66., Conclusion: Clinical tumor size, depth of invasion, and grading of the invasive tumor front were the main predictors of prognosis in patients with stage IB squamous cell cancer of the cervix., (Copyright 1999 Academic Press.)

Objectives: The aim of the study was to evaluate the histological outcome of laser ring biopsies in patients with one or more smears showing cytological mild dyskaryosis where punch biopsies were not performed. The possibilities of predicting histological findings of CIN 2 and CIN 3 were analyzed., Methods: The study comprised 190 laser ring biopsied patients with mildly dyskaryotic smears. Eighty-five patients were excluded from the initial 275 because of up- and downgrading of smears. Conization specimens and smears were reviewed by an experienced histopathologist and two experienced cytotechnicians. The histological findings were correlated to smear findings., Results: Histologically, CIN 2 was found in 60 and CIN 3 in 45 patients (31 and 24%, respectively). The incidence of histological CIN 3 was higher in those with two or more mildly dyskaryotic smears and in those dyskaryotic smears lacking koilocytic change. Mildly dyskaryotic smears without endocervical cells indicated higher incidence of histological CIN 3. The fact that 36 of 96 CIN 2 and 3 lesions (38%) involved only 3 mm or less of the mucosa might be one important explanation for the absence of representative cells in the corresponding smears., Conclusions: Our study has shown that a mildly dyskaryotic smear is a strong indicator of CIN 2-3 histologically, especially in those cases without koilocytic cells and in those with repeatedly abnormal smears. The importance of inadequate sampling is indicated by higher incidence of histological CIN 3 in those with less than optimal smears. These findings are some of the factors to be taken into account when planning treatment strategies for this group of patients.

Granulosa and theca cell tumors of the ovary account for 2-3% of ovarian malignancies. This study includes 54 patients with the diagnosis of granulosa cell tumors of the ovary treated between 1953 and 1987. Median age at diagnosis was 57 (27-83) years. The lesions were staged according to FIGO. The number of patients in various stages was IA, 41; IB, 3; IC, 3; IIB, 6; and III, 1. Median tumor size, 11 cm; range, 0.5-30 cm. Post-menopausal bleeding was diagnosed in 48%, MHC in 37%, proliferative endometrium in 32%, and atypia of endometrial cells in 13% of the cases. Fifty patients were treated with primary surgery, 48 patients were treated with adjuvant external radiotherapy, and 3 patients received complementary chemotherapy. The survival rates in stage I were 94 and 88% after 5 and 10 years, respectively, and in stages II-III were 44% after 5 and 10 years. Overall survival was 90% at 5 years. The frequency of observed mitosis influenced the survival rate: with less or equal 4/10 HPF the survival was 100% in 5 years, with 5-9/10 HPF the survival was 80% in 5 years with a median survival time of 9 years, and with more or equal 10/10 HPF the longest survival was 4 years. At the end of the study, 45 patients (83%) are alive with no evidence of disease, 1 patient is alive with disease, 4 patients are dead of recurrent disease, and 4 patients are dead from intercurrent disease. Endometrial carcinoma was detected in 5 patients. The total survival is better than that with epithelial ovarian cancer as the hormonal symptoms make an early diagnosis possible. Stage for stage the survival is equal. There is an increased incidence of endometrial carcinoma and concomitant other malignancies. The mitotic rate is a well-defined parameter and influences the survival significantly and should be considered the most important prognostic factor at treatment planning.

In a retrospective analysis of 106 cases of endometrial carcinoma stages I-IV (FIGO), the prognostic value of DNA ploidy and nuclear morphometry of tumor cells was evaluated and compared with that of conventional clinical and histopathologic parameters. Paraffin-embedded tumor tissue from the original curettage specimens was used. A flow cytometric technique was employed to distinguish diploid from aneuploid tumors. It was not possible to estimate S-phase rates by this method. Eight different nucleus-related morphometric parameters were computed from representative tumor regions on the original slides. All histologic specimens were reviewed by on the pathologist and graded according to FIGO; nuclear grade was determined separately. Tumor stage, depth of myometrial infiltration, and nuclear grade were the most important prognostic factors with regard to tumor-related survival. DNA ploidy and nuclear morphometry did not add significant prognostic information that could be used to distinguish high-risk and low-risk populations with endometrial carcinomas. The simple nuclear grading system should be further evaluated in prospective studies and compared with DNA analysis and nuclear morphometry performed on fresh-frozen tissue.

Adenoid cystic carcinoma (ACC) is a slow-growing tumor with a marked tendency for perineural and local invasion. The neoplasm occurs more frequently in the head and neck region and only 39 previously described cases of adenoid cystic carcinoma of Bartholin's gland are found in the world literature. Most authors advocate surgery as the primary treatment. Several cytostatic drugs have been tried but the results have been poor. The benefit of radiotherapy in the treatment of this type of tumor is not yet established. This is a report of five new cases all treated with surgery and postoperative radiotherapy. Three patients had histopathologically proven evidence of residual disease after surgery and were subsequently treated with radiotherapy. They are now living without evidence of disease after 28, 51, and 138 months.

Intracavitary irradiation was administered to 366 patients with endometrial carcinoma stage I by a high dose-rate afterloading method using 60Co sources (bulb technique). In 275 cases hysterectomy and bilateral salpingo-oophorectomy were performed 6 weeks later and in 91 cases dilation and curettage was used to verify tumor eradication. In 58% of the hysterectomy specimens no residual carcinoma was detected at the histopathologic evaluation. In the group treated with radiotherapy alone 74% showed no carcinoma remnants in the curettings 3 months after therapy. The effect of the fractionation dose was evaluated in relation to the outcome of the histopathologic examination. The proportion of specimens with no residual carcinoma increased from 27% for the 5 Gy per fraction group to 78% for the 10 Gy per fraction group. Recurrences were recorded in 13% in the hysterectomy group and in 29% in the group treated with radiotherapy alone. The 5-year corrected survival rate for the combination-treated group was 88% and for the group treated with radiotherapy alone 72%. If the dose per fraction is specified in the range of 5 to 8 Gy the high dose-rate afterloading technique seems safe with a tumor control rate and a frequency of radiation reactions comparable to the manual radium method.

Sheep were prepared with a chronic lung lymph fistula and studied unanesthetized following septicemia by infusion of live Escherichia coli 10(9) ml/kg bw or injection of oleic acid 0.05 ml/kg bw. Extravascular lung water (EVTV) was measured with thermal-dye technique and compared to gravimetrically measured lung water (EVWV). Septic sheep had increased pulmonary artery pressure, reduced mean arterial blood pressure and reduced cardiac output. In control animals there was a correlation between EVTV-EVWV of r = 0.70. In animals given oleic acid lungs were macroscopically edematous and the correlation was r = 0.93. In septic sheep, however, no correlation could be found between EVTV and EVWV (r = -0.25). The thermal-dye technique was found to give erroneously high values. This finding could probably be due to erythrostasis and leukocyte plugging with uneven perfusion and prolonged transit times due to reduced cardiac output.

The effect of preparative trauma on lung microvascular permeability during lung lymph fistula preparation in sheep was investigated. Two groups of anesthetized sheep were used. In group I (controls: n = 3) the lymph fistula was prepared 3 to 4 days before the experiment. In group II (experimental group: n = 13): left atrial and lymph catheters were introduced on the day of the experiment. All animals had catheters in the aorta and pulmonary artery. The stable prostanoid metabolites thromboxane B2 (TxB2) and 6-keto-PGF1 alpha (6-keto) were measured in lymph. In group I systemic (Psa) and pulmonary pressures (Ppa) remained constant. Leukocytes and platelets were unchanged as was pulmonary lymph flow (QL) and the lymph to plasma total protein concentration ratio (L/P). In group II Psa and Ppa remained constant. All sheep developed a temporary leukopenia and thrombocytopenia. TxB2 and 6-keto were low and stable during anesthesia in group I but were elevated immediately following surgery in group II but values normalized during a 4-hr observation period. QL during the first half hour was 1.9 +/- 0.3 ml/30 min and increased over 5 hr to 3.1 +/- 0.7 ml/30 min corresponding to 60 +/- 15% over baseline (P less than 0.01). L/P did not change. These changes could be due either to changing permeability or surface area. These effects of preparative trauma indicated that the model was not in a steady state. This observation should be kept in mind when evaluating studies using the lung lymph fistula in acute experiments.

Uteri from four patients with endometrial adenocarcinoma and concomitant "functioning" endometrium were examined by a whole-organ sectioning technique. All showed one or several "progesterone-refractory" mucosal areas or polyps, mainly in the tubal recesses. In one patient the adenocarcinoma obviously originated in a fundal polyp, and in the others the tumors could have arisen in mucosal areas not adequately responding to progesterone. These findings do not in fact contradict the "unopposed estrogen" theory of the genesis of endometrial carcinoma.

Paraffin-embedded curettage material from 120 patients with stage I and II endometrial cancer were analyzed by flow cytometry. The follow-up time in all cases was at least 5 years or until death. It was possible to determine DNA ploidy in 111 cases and S-phase was also evaluated in 92/111 cases. DNA ploidy and S-phase were then compared with the FIGO grading and clinical outcome. DNA ploidy results showed that 11% of grade 1 tumors were aneuploid, 14% of grade 2, 42% of grade 3, and 85% of the cases of uterine papillary serous carcinoma (UPSC) were aneuploid. Patients with tumors that showed an S-phase fraction below 5% had a cumulative 5-year cancer mortality of 7% whereas 49% of the patients with tumors having S-phase fraction above 10% died of their cancer. The prognostic significance of DNA and S-phase correlated fairly well with the FIGO grading as determined by a pathologist with special interest in gynecologic oncology and the DNA parameters added prognostic information independent of the FIGO grading.