Nuclear DNA content, proliferative activity, and p53 expression related to clinical and histopathologic features in endometrial carcinoma.

The purpose of this study was to evaluate the prognostic impact of image cytometry DNA ploidy, MIB-1, and p53 in relation to clinicopathologic variables in 376 consecutive patients with endometrial carcinoma stages I-IV. Following primary treatment 358 patients were considered tumor-free. Relapses and tumor-specific deaths of these patients were noted. Image cytometry DNA ploidy (n = 340) and expression of MIB-1 (n = 318) and p53 (n = 323) were studied. In univariate analysis, stage (P < 0.001), histopathologic subtype (P < 0.001), degree of differentiation (P < 0.001), HRT (P = 0.034), DNA ploidy (P < 0.001), and p53 (P < 0.001) were significant predictors of relapse. Patient age showed that the estimated mean risk of relapse increases with nearly 64% per decade in life (P 0.003), and the MIB-1 expression with 21% per 10-unit increment (P 0.004). In multivariate analysis, degree of differentiation, MIB-1, and p53 lost their prognostic capability. However, after stage and histopathologic subtype, image cytometry DNA ploidy was the strongest predictor of outcome and was of value in predicting the risk for relapse. The combination of DNA ploidy, MIB-1, and p53 expression was an even stronger predictor of relapse-free survival than the individual prognostic factors.

Human papillomavirus infection, centrosome aberration, and genetic stability in cervical lesions.

DNA replication and centrosome duplication have to be strictly synchronized to guarantee genomic stability. p53, pRb, cyclin E, and cyclin A are reported to be involved in the synchronizing process. We investigated the relationship between papillomavirus infection, centrosome aberration and aneuploidy during genesis of cervical carcinoma. The number of centrosomes found in cells from normal cervical epithelium (n = 5), condyloma acuminata (n = 5), cervical intraepithelial neoplasia (CIN) I, II, and III (n = 14) and invasive cervical carcinoma (n = 5) was analyzed by gamma tubulin immunofluorescence staining. The nuclear DNA content was investigated by image cytometry and human papillomavirus (HPV) infection was determined by polymerase chain reaction. Normal epithelia and condyloma acuminata showed cells with one or two centrosomes, whereas CIN lesions showed cells with an increasing number of centrosomes. This abnormality was found to be lowest in CIN I lesions, increased with advancing grade of CIN and was highest in lesions of invasive carcinomas. In parallel, an increasing number of cells with aberrant DNA content was seen. All carcinomas and all except one of the CIN III lesions showed aneuploidy. Three CIN II cases were aneuploid and two cases with CIN I were tetraploid. Normal epithelia and condyloma acuminata showed diploidy. All invasive carcinomas and lesions with CIN were positive for high-risk HPV types 16, 18, or 31, except one invasive carcinoma and one CIN II lesion positive for universal primers only. Three condyloma acuminata were HPV 16-positive and one HPV 6-positive. The results suggest that high-risk HPV infection is correlated to a progressive numerical disturbance of centrosome replication followed by progressive chromosomal aberrations in CIN lesions and invasive carcinomas.

A population-based five-year follow-up study of cervical human papillomavirus infection.

OBJECTIVE: The purpose of this study was to determine the long-term tendency for cervical human papillomavirus infections to persist in the general population. STUDY DESIGN: From 500 women who participated in a 1991 population-based survey, 90 healthy women with normal results of cytologic examination (women with human papillomavirus deoxyribonucleic acid detected and age-matched control women without human papillomavirus deoxyribonucleic acid detected) were interviewed and examined 5 years later colposcopically, cytologically, and with human papillomavirus serologic testing and human papillomavirus deoxyribonucleic acid testing by polymerase chain reaction with 2 different consensus primer pairs (MY09 and MY11 and GP5(+) and GP6(+)), type-specific polymerase chain reaction, and deoxyribonucleic acid sequencing. RESULTS: The 5-year human papillomavirus clearance rate was 92%. Only human papillomavirus type 16 infections persisted. Colposcopic impression of grade 2 cervical intraepithelial neoplasia was associated with persistent human papillomavirus 16 infection (P <.03). Human papillomavirus detection was associated with sexual history. Human papillomavirus type was the only determinant of human papillomavirus persistence. CONCLUSION: The high clearance rates in a population-based setting with a 5-year follow-up period imply that inclusion of human papillomavirus deoxyribonucleic acid testing in population-based cervical screening programs should target persistent infection.

Classification of human ovarian tumors using multivariate data analysis of polypeptide expression patterns.

Large amounts of data on quantitative gene expression are generated by procedures such as 2-DE analysis of proteins or cDNA microarrays. Quantitative molecular variation may potentially be used for the development of methods for the classification of tumors. We used here the statistical concepts of principal components analysis (PCA) and partial least square analysis (PLS) in an attempt to type ovarian tumors. Using a set of 170 polypeptides, 22 tumors were used to establish a model ("learning set") for classification into 3 groups (benign/borderline/malignant). Eighteen tumors were then used to test the model. Six of 8 carcinomas and 3 of 4 borderline tumors were correctly classified. Two of 6 benign lesions were correctly classified, 3 were classified as borderline and 1 as carcinoma. We conclude that it may be possible to classify tumors according to their constitutive protein expression profile using multivariate analysis, thus making classification by artificial intelligence a future possibility.

Laminin-5 as a marker of invasiveness in cervical lesions.

BACKGROUND: Treatment decisions for cervical cancer, a common disease worldwide, depend on demonstrating whether or not tumor invasion of the surrounding tissue has occurred. Invasion can be difficult to assess by standard histopathologic methods, especially when limited amounts of tissue are available. Several studies of a variety of cancers have reported increased expression of laminin-5-an important attachment protein for epithelial cells-in invasive carcinomas. This study was designed to investigate whether the presence of laminin-5 is related to the invasive capacity of cervical lesions. METHODS: We used immunohistochemical methods to stain archival, paraffin-embedded sections of cervical lesions with a polyclonal antibody specifically targeting the gamma2 chain of human laminin-5 protein. The study sample included 23 lesions of mild and moderate dysplasia (cervical intraepithelial neoplasia [CIN] 1 and 2, respectively), 32 lesions of severe dysplasia or carcinoma in situ (CIN 3), 15 lesions of microinvasive cancer, and 20 lesions of frankly invasive cancer. Cellular proliferative activity was also investigated by the use of monoclonal MIB-1 (directed against the antigen Ki-67) and anticyclin A antibodies. RESULTS: Invasiveness of cervical lesions was positively associated with immunohistochemical staining of the gamma2 chain of laminin-5 (two-sided P =.001). All CIN 1 and CIN 2 lesions-except one CIN 2 lesion later shown to be invasive cancer-and 21 CIN 3 lesions tested negative for the gamma2 chain of laminin-5. Eleven CIN 3 lesions and all invasive cancers tested positive for this protein. One lymph node metastasis and a pleural metastasis from one of the patients with invasive cancer showed strong immunohistochemical positivity. Proliferative activity increased with advancement of the lesion but was not confined to cells positive for the gamma2 chain of laminin-5. CONCLUSIONS: These data suggest that antibodies directed against the gamma2 chain of laminin-5 can identify cervical lesions with invasive capacity and thus may be useful as a sensitive marker of early invasion.

Two-dimensional gel analysis of protein expression in ovarian tumors shows a low degree of intratumoral heterogeneity.

The process of tumor progression leads to the emergence of multiple clones, and to the development of tumor heterogeneity. One approach to the study of the extent of such heterogeneity is to examine the expression of marker proteins in different tumor areas. Two-dimensional gel electrophoresis (2-DE) is a powerful tool for such studies, since the expression of a large number of polypeptide markers can be evaluated. In the present study, tumor cells were prepared from human ovarian tumors and analyzed by 2-DE and PDQUEST. As judged from the analysis of two different areas in each of nine ovarian tumors, the intratumoral variation in protein expression was low. In contrast, large differences were observed when the protein profiles of different tumors were compared. The differences in gene expression between pairs of malignant carcinomas were slightly larger than the differences observed between pairs of benign tumors. We conclude that 2-DE analysis of intratumoral heterogeneity in ovarian cancer tissue indicates a low degree of heterogeneity.

A multicenter study comparing two endometrial sampling devices--Medscand Endorette and Pipelle de Cornier.

BACKGROUND: To compare two endometrial sampling devices Medscand Endorette and Pipelle de Cornier with respect to tissue collecting ability, diagnostic accuracy and side effects. METHODS: A prospective, multi-center, cross-over study in 152 women with a medical indication for endometrial biopsy. Samples were collected from each patient on the same occasion with both devices, the order of which was randomized. Statistical analysis was based on pairwise comparison in contingency tables with McNemars chi2 test. RESULTS: One hundred and forty-five of 152 (95%) women were successfully sampled. There was no difference between the devices concerning discomfort and bleeding. However, Medscand Endorette seemed to have a higher capacity than Pipelle for collecting an adequate sample. This difference was noted when the two devices were compared in the position as first instrument as well as when results were compared in the main target group for this type of examination, for women over 55 years. CONCLUSIONS: The new Medscand Endorette was preferred due to its higher capacity for collecting adequate samples.

Phenotypic analysis of ovarian carcinoma: polypeptide expression in benign, borderline and malignant tumors.

Studies of multiple markers in tumors are required for adequate biological characterization. We have characterized the expression of multiple proteins in human ovarian tumors using the technique of 2-dimensional gel electrophoresis (2-DE/PDQUEST). Tumor cells were prepared from the tissue of 22 ovarian tumors. Large variations were observed between tumors in the expression of various polypeptides, indicating heterogeneity in gene expression. An increase in the spot density of 2 cell-cycle-related proteins, PCNA and OP18/stathmin, was observed in carcinomas. Borderline tumors expressed low levels of these proteins. Significant increases in the levels of nm23, GST-pi, elongation factor 2 and triose phosphate isomerase were recorded in ovarian carcinomas. Furthermore, decreases in the levels of tropomyosin-2 and lamin C were observed in malignant as compared with benign tumors. The pattern of expression of 9 protein markers was examined in individual tumors. All malignant tumors showed simultaneous alterations in the expression of 5 or more of these proteins, whereas no benign tumor showed alterations in the expression of more than 3 polypeptides. Borderline tumors showed alterations in 0 to 6 markers. We conclude that the simultaneous analysis of multiple polypeptides, which can be achieved by 2-DE, is useful for characterization of gene expression and diagnostic studies in ovarian tumors.

Aggressive endometrial cancer in a young patient.

An unusually aggressive case of endometrial cancer in a 30 year old woman is presented. The patient experienced abnormal uterine bleeding, at times requiring blood transfusions, for almost half a year before the diagnosis was revealed. For obvious reasons there is a reluctancy to perform invasive examinations in young women. The diagnostic options are discussed.

Tamoxifen-induced DNA adducts in endometrial samples from breast cancer patients.

Tamoxifen-induced DNA adducts were analyzed with the (32)P-postlabeling method using high-performance liquid chromatography (HPLC)-radioactivity detection from endometrial tissue of breast cancer patients and controls. Liver DNA from tamoxifen-treated rats was used as a positive standard. In blind analysis, five of the seven samples from tamoxifen-treated patients showed DNA adducts; none of the five controls were positive. The identity of the tamoxifen adduct was confirmed by using different chromatographic systems, isolating the HPLC fractions and running them on TLC, with or without spiked rat liver samples. The level of adducts in the treated patients was 2.7 adducts/10(9) nucleotides in the HPLC analysis.

Premature rupture of the membranes (PROM) at term in nulliparous women with a ripe cervix. A randomized trial of 12 or 24 hours of expectant management.

OBJECTIVE: To compare maternal and neonatal outcomes after 12 or 24 hours of expectant management in healthy nulliparous women with a ripe cervix and PROM at term. DESIGN: A prospective, randomized study. LOCATION: Karolinska Hospital, Stockholm, Sweden. SUBJECTS: Two hundred and five healthy nulliparous women with singleton pregnancies, cephalic presentation, gestational duration 36 to 42 weeks, randomized to 12 or 24 hours of expectant management after evaluation of the cervical score (> 5). If spontaneous labor did not occur, induction was performed with oxytocin after 12 or 24 hours, respectively. MAIN PARAMETERS: Maternal early morbidity and neonatal infections, obstetric intervention rate (cesarean section or instrumental delivery). RESULTS: The cesarean section rate was 4% in each group. The vacuum extraction rate was 21% in each group. Induction of labor was performed in 47% of the women allocated to 12 hours of expectant management vs 17% of the women allocated to 24 hours of expectant management (p < 0.05). The maternal morbidity rate was almost negligible. Only a few fetal infections occurred and no difference was noted between the groups. CONCLUSIONS: In healthy nulliparous women at term with a ripe cervix, expectant management over 24 hours vs 12 hours resulted in fewer inductions of labor and no increase in instrumental deliveries, without any increase in neonatal or maternal morbidity.

HPV detection in cytological cases with condylomatous or dysplastic changes: a study with PCR and in situ hybridization on cytological material.

Cytobrush samples of 80 patients, who previously had a cytological or histopathological diagnosis of condyloma and/or dysplasia were investigated for human papillomavirus infection (HPV) by polymerase chain reaction (PCR) and in situ DNA hybridization technique (ISH). The results were compared with concomitantly obtained cytological Pap-stained smears or, in some cases, histological sections. The time between the diagnosis of the original and the concomitant cytology/histopathology was less than 1 yr. Six additional patients had similar morphological diagnoses 2-4 yr before. Five more cases were included on clinical diagnosis of HPV. Compared with the original morphological diagnoses, 70% of the cases were positive by PCR and/or ISH. The concomitant morphology was not diagnostic of HPV in 44 out of 80 cases (55%), showing a relatively high percentage of cases morphologically normalized in the interval since the first specimen was taken. After detection with PCR, 30 cases (37.5%) were negative for HPV. Only one of the patients with a previous disease 2-4 yr before was HPV positive by PCR and two out of five patients with a clinical diagnosis of HPV. ISH could be performed on 67/80 cases, 43 of which were positive for HPV. There was a good agreement between the results of ISH and PCR, but there were six cases positive by ISH and negative by PCR. In these cases, few infected cells may have escaped detection by PCR. Both methods seem to be able to detect silent HPV infections and comparison with concomitant cytology/histopathology shows that morphology alone is insufficient for HPV detection in these cases.

Human papillomavirus in cell samples from Stockholm Gynecologic Health Screening.

We compared the results of cytologic screening of 500 women in the Stockholm Gynecologic Health Control with human papillomavirus (HPV) detection by polymerase chain reaction (PCR) and in situ hybridization (ISH). There were two main age groups, one 30 years and younger and the other 40 years and older. There were relatively more women with HPV infection in the younger group than in the older one (15.7% as compared to 11.1%), but the difference was not significant in our material. Most cases (8/12) with cytologic atypia were HPV positive by PCR. HPV type 16 was most common, followed by types 31 and 18. HPV of unknown types was detected in 43.7% of HPV-positive cases. There was excellent agreement between PCR and ISH in detecting and typing HPV.

Value of uterine artery Doppler in endometrial cancer.

Twenty-seven women with endometrial cancer were studied with Doppler ultrasound coupled with a vaginal probe. Pulsatility index of the flow velocity of the uterine artery was recorded and compared to that of a control group. The subjects and the controls did not differ in blood flow measurements. There was no correlation between severity of disease and flow velocimetry values. Eleven of the patients underwent brachytherapy prior to surgery. Administration of brachytherapy resulted in a decrease of the peripheral resistance. The results of this study indicate that Doppler velocimetry of the uterine artery is not a valuable tool in discriminating between malignant and benign endometrium.

DNA-content in endometrial carcinoma of tamoxifen-treated breast cancer patients.

DNA measurements and histopathologic evaluation were performed in 17 patients treated with adjuvant tamoxifen for early breast cancer and who developed endometrial carcinoma during or after the tamoxifen therapy. The tumors were exclusively characterized by euploid DNA content except for two cases, one mixed mesodermal sarcoma, a highly malignant and rare tumor, and one adenocarcinoma. Although the use of adjuvant tamoxifen therapy most likely enhances the risk of developing endometrial carcinoma, the beneficial effects of adjuvant breast cancer treatment is of well-known clinical importance. The hazards of giving long-term tamoxifen seem to be low since the endometrial tumors were associated with low-grade malignancy and euploid DNA pattern.

Descriptive clinicopathologic study of 17 patients with endometrial cancer during or after adjuvant tamoxifen in early breast cancer.

BACKGROUND: Studies have shown that patients with early-stage endometrial cancer who have previously used endogenous estrogen (oral contraceptives or estrogen replacement therapy) have a favorable prognosis. This has not yet been demonstrated for patients with early-stage endometrial cancer who have received tamoxifen. In addition, studies have raised the question of whether women receiving tamoxifen are at increased risk of endometrial cancer. PURPOSE: Our aim was to determine whether the prognosis is favorable for patients with diagnosis of endometrial cancer after adjuvant treatment with tamoxifen for breast cancer. METHODS: We matched 931 patients from the Stockholm Adjuvant Tamoxifen Trial in early breast cancer against the Swedish Cancer Registry and identified 17 who subsequently had endometrial cancer. These patients had been randomly assigned to receive 40 mg/d tamoxifen orally for 2 years beginning 4 weeks after surgery for breast cancer. Histologic specimens, patient records, and death certificates were reviewed to verify treatment and causes of death. RESULTS: Thirteen of the 17 patients diagnosed with endometrial cancer were alive; for three of the four who had died, the cause of death was endometrial cancer. All 16 evaluable tumors except one were World Health Organization (WHO) histologic grades I-II. Only one patient had advanced disease (stage IV); the remaining tumor was a mixed mesodermal malignant tumor that could not be classified under the WHO grading system. Median time for adjuvant tamoxifen use was 24 months (range, 6-60 months) with a median cumulative tamoxifen dose of 29 g (range, 7-72 g). Median time from initiation of adjuvant tamoxifen to diagnosis of endometrial cancer was 32 months (range, 6-130 months). Ten-year actuarial survival after diagnosis of endometrial cancer for the 17 patients treated with tamoxifen was 73%. CONCLUSION: Because of the small number of patients, our results do not rule out the possibility of a favorable prognosis for patients with a diagnosis of endometrial cancer following tamoxifen treatment. IMPLICATIONS: The incidence of secondary endometrial cancer reported in this study following treatment of breast cancer patients with tamoxifen at doses of 40 mg/d in a large clinical trial is higher than that reported for previous large trials of tamoxifen at doses of 20 mg/d. Thus, tamoxifen dosage may be a critical factor in the subsequent occurrence of endometrial cancer. Our results also suggest two important considerations for improved follow-up in long-term tamoxifen trials: careful registration of second cancers and routine gynecologic examinations to ensure early detection of endometrial cancer.

Methodological aspects on cytochemical DNA assessment of adenocarcinoma of the endometrium by means of image and flow cytometry using conventionally formalin-fixed and paraffin-embedded specimens.

Sometimes widely diverging results have been reported as regards the nuclear DNA ploidy pattern of adenocarcinomas of the endometrium. Since such discrepancies might be due to differences in the techniques applied, it seemed worthwhile to investigate this possibility in conventional uterine curetted specimens. In order to obtain a high incidence of tumours with cancer cell nuclei showing "aneuploid" DNA distribution pattern, a selection was made, so that only those adenocarcinomas that had led to a fetal outcome of the neoplastic disease were examined. The results of two image cytometric (ICM) techniques for cytochemical nuclear DNA assessments were compared. One was direct photographic cytometric measurements on Feulgen-stained sections; the other was densitometric assessments on isolated tumour cell nuclei of deparaffinised and disintegrated specimens. In 39 cases out of 43 the DNA ploidy pattern was the same by means of the two techniques. However, about half the numbers of the specimens (40 out of 83) were lost during the deparaffinisation and disintegration procedure. As far as could be found from a limited study on 20 (out of the 43) selected cases, these losses of specimens became even greater when the flow-cytometric (FCM) technique was applied on the deparaffinised specimens; about one third of these specimens were not possible to evaluate. In addition, in those where assessments by means of FCM could be made, the DNA ploidy pattern obtained differed from that of the two ICM techniques in not less than 80% of the cases. Broad peaks and high amounts of counts in the background in the DNA histograms indicated that most of the DNA assessments made by means of FCM on archival material of the present kind of curetted specimens of endometrial adenocarcinomas gave no reliable results. Consequently, differences in the techniques applied in cytochemical assessments of the nuclear DNA distribution pattern in endometrial carcinomas can explain the more or less controversial results reported from different laboratories.

The prognostic significance of growth pattern and its relation to tumor cell nuclear DNA content in endometrial carcinoma.

Hysterectomy specimens from 21 endometrial carcinoma patients, who died from their disease, and 23 patients selected at random from 307 survivors, were analysed for tumor growth pattern and tumor cell nuclear DNA content. The results indicate that tumor growth pattern, reflected by the mode of infiltration, is significantly correlated to the clinical course of the disease. Patients with carcinomas exhibiting contiguous growth pattern had a better outcome than patients with discontiguously growing carcinomas. It was also found that tumor growth pattern correlated well with tumor nuclear DNA content. It is suggested that the pattern of infiltration of the tumors is a sensitive predictor of prognosis and that this prognostic information, which only can be obtained postoperatively, to a large extent is reflected by tumor cell nuclear DNA content in curetted diagnostic material, obtained prior to treatment.

DNA and nuclear protein characteristics in non-neoplastic and neoplastic endometrial tissue.

Feulgen-DNA and nuclear protein (NP) measurements were performed on non-neoplastic and neoplastic endometrium. Non-neoplastic endometrial cells were exclusively characterized by euploid nuclear DNA content. The NP content may vary significantly in relation to the proliferative stage as reflected by a 2-3-fold increase NP/DNA ratio in growing as compared to growth arrested cells. Endometrial adenocarcinomas could be subdivided into euploid and aneuploid types. The euploid tumors were found to exhibit DNA and NP characteristics comparable with those of normal tissue. In contrast, aneuploid tumors showed DNA and NP characteristics indicating increased proliferative activity as well as a pronounced disorder between the DNA and protein cycle.

The effect of labetalol on contractility of human myometrial preparations.

Because of results in animal experiments which demonstrated a partial beta 2-adrenoceptor activity of labetalol on rat uterine smooth muscle the present study was conducted in human preparations. The following results were obtained: 1. Rhythmic uterine contractions with a defined steady-state amplitude and frequency were elicited spontaneously and after methylergometrine. 2. Labetalol reduced amplitude of contractions dose-dependently after 3 h of incubation. Frequency was unaffected. 3. The tocolytic effect of labetalol is apparent only at high concentrations, above those used in the treatment of hypertension. 4. Neither beta 2-specific adrenoceptor blockade with ICI 118,551 nor alpha-blockade with phentolamine changed amplitude of contraction, either alone or in combination with labetalol. 5. Labetalol has little tocolytic effect on human myometrium in vitro. This effect is unrelated to alpha- or beta-antagonism, but seems to depend on a direct smooth muscle depressant effect. In conclusion, from the present in vitro experiments using human myometrial preparations, it seems unlikely that labetalol would interfere with the normal process of labor when used for the treatment of pregnancy hypertension.