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The effect of ovarian cancer screening on survival is enabled by women who continue to actively participate in screening. In this report, factors that affect participation are examined. Participants included 13,963 Kentucky women who received 56,392 screens accounting for a 46,113 screening year experience. Background, health history and reasons for participating in transvaginal sonographic screening were collected via a self-reported questionnaire. Screening participants were characterized as > 50 years old, mostly married, well educated, medically insured, health conscious women, living in the vicinity of the screening centre or 51-150 miles away. Approximately 70% of the participants can be considered active in the study based upon a screening visit during the 1997-1998 2-year period. The probabilities of a return screen at 1, 2, 5 and 7.5 years were 77.8%, 72.0%, 58.7% and 50.6%, respectively. A total of 96% of return visits occurred within 2 years, with 33.7% having intervals of < 1 year. Perceived family history was not observed to affect continuation. However, abnormal findings were associated with a shortened participation. These high levels of continuation in ultrasound screening indicate that women take this disease seriously and demonstrate that this disease is of consequence to them.

From December 1987 to December 1993, 6470 women underwent screening with transvaginal sonography (TVS) as part of the University of Kentucky Ovarian Cancer Screening Project. Two groups of women were eligible to participate in this investigation: (i) asymptomatic postmenopausal patients or patients >50 years of age, and (ii) asymptomatic women >30 years of age with a family history of ovarian cancer. Ovarian volume was calculated using the prolate ellipsoid formula (length x height x width x 0.523). An abnormal sonogram was defined by (1) an ovarian volume >10 cm3 in postmenopausal women or >20 cm3 in premenopausal women, and (2) a papillary or complex tissue projection into a cystic ovarian tumor. All women with an abnormal TVS had a repeat sonogram in 4-6 weeks. Patients with persistently abnormal scans had a serum CA-125 determination, tumor morphology indexing, and color Doppler sonography. Ninety patients (1.4%) with a persisting abnormality on TVS underwent exploratory laparotomy or laparoscopy for tumor removal. Thirty-seven patients had serous cystadenomas and six had primary ovarian cancers. Five patients had Stage IA ovarian cancer and one patient had Stage IIIB disease. Only one of the ovarian cancer patients had a palpable abnormality on pelvic examination, and none had an elevated (>35 u/ml) serum CA-125. All these patients are presently alive and well 1-5 years after conventional therapy. There was one false negative in this study, a 38-year-old white female who was noted to have a small ovarian cancer at the time of laparoscopic prophylactic oophorectomy 11 months after a normal scan. Over 17,000 screening years have been accrued and there have been no deaths from primary ovarian cancer in the screened population. A cost analysis of TVS screening is presented.

Obtaining a high quality nucleic acid or protein gel may be an easier and less daunting task than labeling the gel for publication. Techniques for adding information and identifiers to gels range from laboriously applying lettering or labels on gel photographs to computerized approaches that work directly with digitized gel images and produce photographic-quality output. The computerized approach may require specialized software and a considerable investment in a photographic-quality output device, like a dye sublimation printer. It can be argued that electronically processed gel images do not actually achieve the quality readily obtained photographically. We describe an approach that uses photographic gel images that are quickly and easily labeled using the drawing-lettering capability of a commercial word processor in widespread use (Microsoft Word for Windows Version 6).

Background: The three most extensively evaluated screening methods for ovarian cancer are pelvic examination, serum CA 125, and transvaginal sonography (TVS). The lack of sensitivity of pelvic examination and serum CA 125 has limited their use in ovarian cancer screening. Currently, the most effective screening method for ovarian cancer is TVS., Methods: Transvaginal sonography was performed with a standard ultrasound unit and a 5.0 MHz vaginal transducer. Each ovary was measured in three dimensions and ovarian volume was calculated using the prolate ellipsoid formula (L x H x W x 0.523). An ovarian volume greater than or equal to 20 cm3 in premenopausal women and greater than or equal to 10 cm3 in postmenopausal women was considered abnormal. Also, any internal papillary projection from the tumor wall was considered abnormal. A patient with an abnormal screen had a repeat TVS in 4-6 weeks. Women with a persisting abnormality on TVS underwent pelvic examination, serum CA 125 determination, Doppler flow sonography, and tumor morphologic indexing before operative tumor removal., Results: Eighty-five hundred asymptomatic women underwent TVS screening. One hundred twenty-one of these women had a persisting abnormality and underwent tumor removal. Fifty-seven patients had serous cystadenomas and eight had primary ovarian cancers. Six patients had Stage IA ovarian cancer, one had Stage IIC ovarian cancer, and one had Stage IIIB ovarian cancer. Only one of these patients had palpable ovarian enlargement on clinical examination and one had an elevated serum CA 125. All patients are alive and well 4-61 months after conventional therapy. The direct cost of TVS screening was highest during the initial years of the program and fell progressively to $30/screen during the 4th year of the study. Worldwide, more than 14,000 women have been screened using ultrasonography, and 19 ovarian cancers have been detected. More than 20,000 patient-screening-years have been accrued, and there have been no deaths from primary ovarian cancer in the screened population., Conclusions: Transvaginal sonography screening causes a decrease in stage at detection and a decrease in case-specific mortality. Further study is needed to determine if annual TVS screening will significantly reduce ovarian cancer mortality. The cost for TVS screening is reasonable and is well within the range of that reported for other screening tests.

The objective was to identify factors influencing participation in screening for ovarian cancer using transvaginal sonography in the free experimental program at the University of Kentucky over its 6+ year history. Database records for screenings, performed from 1987 to June 1994, were utilized. Computer sorts, 1990 census information as predictors, and stepwise multiple regression analysis were employed. Participation in the model ovarian screening program took 3-4 years to approach > 300 screens/month, with repeat screenings exceeding new subject participation in this time period. A number of participants traveled > 200 miles for screening on both initial and repeat encounters. Analysis of distance to the screening site, median family income, county physician population, and education levels indicated that distance and then education correlated best with participation. Unit screening cost shrank from $45 to under $25 when maximal participation was achieved. Distance and education correlated with participation. Expenses compare favorably with diagnostic procedures for other diseases.

Objective: To examine the suitability of estradiol labeled with iodine I 123 at 16 alpha for imaging estrogen-receptor-positive breast carcinoma using imaging instrumentation that is widely available., Design: Single-photon emission computed tomographic imaging survey of 29 women with suspected primary or expected recurrent breast carcinoma., Setting: University-based referral center., Participants: Twenty-nine women undergoing diagnosis for primary or recurrent breast carcinoma. Selection was voluntary., Main Outcome Measure: Qualitative imaging study designed to provide tomographic data of radioligand retention and descriptive data of imaging results., Results: Single-photon emission computed tomographic imaging using 123I-estradiol at 16 alpha was performed for patients with breast carcinoma. Independent readers, without knowledge of receptor status or proven disease, interpreted the films. Scintigraphic detection was most noteworthy in patients with chest wall tumors and inflammatory breast cancer. Agreement between readers was 98% for true-negative readings and 94% for true-positive readings, but only 60% for false-positive and false-negative film readings., Conclusions: Our results indicated that areas shown on imaging were also found to have estrogen-receptor activity and that radioligand accumulation can occur with low frequency in some surgically explored tissue. Radioligand imaging with 16 alpha-123I-estradiol can locate estrogen-receptor-positive breast tumors, including some that may be difficult to detect using conventional diagnostic imaging.

From 1987 to 1992, 3220 asymptomatic postmenopausal women underwent screening with transvaginal sonography (TVS) as part of the University of Kentucky Ovarian Cancer Screening Project. Ovarian volume was calculated using the prolate ellipsoid formula (length x height x width x 0.523). An abnormal sonogram was defined by (1) an ovarian volume > 10cm3 or (2) a papillary projection into a cystic ovarian tumor. All women with an abnormal TVS had a repeat sonogram in 4-6 weeks. If the repeat sonogram was abnormal, a morphology index score was assigned to each tumor, and a serum CA-125 was obtained. The patient then had a pelvic examination and an exploratory laparotomy. Forty-four patients (1.4%) with a persisting abnormality on TVS underwent exploratory laparotomy. Twenty-one patients had serous cystadenomas and 3 had primary ovarian cancers. Two patients with primary ovarian cancer had Stage IA disease and one had Stage IIIB disease. All patients with ovarian cancer had normal pelvic examinations and normal serum CA-125 levels, and are presently alive and well 32, 31, and 8 months after conventional therapy. Over 5000 screening years have been accumulated at this institution, and there have been no ovarian cancer deaths in the screened population. TVS screening has produced a decrease in stage at detection and case-specific mortality from ovarian cancer. A multi-institutional trial to test the efficacy of TVS as a screening method for ovarian cancer is indicated.

A morphology index based on morphologic characteristics of ovarian tumors was developed. Specific categories included tumor volume, wall structure, and septal structure. A point scale (0-4) was developed within each category with the total points per evaluation varying from 0-12. Sonograms on 121 patients undergoing exploratory laparotomy for ovarian masses were evaluated using this index. Eighty ovarian tumors had a morphology index score < 5, and all were benign (negative predictive value, 1.000). In postmenopausal patients, a morphology index score > or = 5 had a positive predictive value for malignancy of 0.45. All ovarian malignancies had significant abnormalities in wall structure and all had a total volume in excess of 10 cm3. The findings of the present investigation indicate that the morphology index is a cost effective adjuvant method which significantly increases the specificity and positive predictive value of transvaginal sonography. The routine application of a morphology index to screening sonography should decrease the amount of diagnostic surgery performed in order to detect each case of ovarian cancer.

From 1981-1991, 15 patients with primary fallopian tube carcinomas were treated at the University of Kentucky Medical Center. Immunohistochemical staining for CA-125 was performed on tumor specimens from all cases. Thirteen tumors (87%) stained positively for CA-125. Antigen staining was most intense in the apical portions of carcinoma cells. Serum CA-125 levels were measured in 5 patients and were elevated in 4 (80%). There was a positive correlation between tumor and serum antigen expression in these cases. Serum CA-125 levels accurately reflected disease status in the patients studied. These data suggest that CA-125 is a useful marker in patients with fallopian tube carcinoma. Immunohistochemical localization of CA-125 in tumor tissue should predict which patients will benefit most from serial antigen determinations.

Background: Despite advances in evaluation and treatment, ovarian cancer mortality has decreased minimally during the past two decades. Most patients have advanced-stage disease at diagnosis, and the prognosis is poor. As a result, there has been increasing interest in the development of methods for the early detection of ovarian cancer. To benefit from screening, a disease should (1) be a significant cause of mortality, (2) have a high prevalence in the screened population, (3) have a preclinical phase that can be detected by screening, and (4) be amenable to therapy, such that the survival rate of patients with early-stage disease is significantly higher than that of patients with advanced-stage disease. Ovarian cancer fulfils all of these criteria., Methods: An optimal screening method should be safe, easy to do, time efficient, and acceptable to the patients being screened. Most importantly, it should have a high sensitivity and specificity., Results: Currently, the most effective screening methods for ovarian cancer are serum CA 125 levels and transvaginal sonography (TVS). In screening studies, the serum CA 125 level has had a reasonably high specificity but a low sensitivity. Currently, approximately 8000 asymptomatic women have been screened with TVS. Ten primary ovarian cancers were detected, and all were Stage I lesions. Patients whose tumors were detected by TVS all have been cured by conventional treatment. TVS screening has resulted in a significant reduction in stage at detection and in the case-specific death rate from ovarian cancer. In these studies, TVS has had a high sensitivity but only a moderate specificity. CA 125 level, Doppler flow sonography, and the use of a morphology index are being evaluated as methods to increase the specificity of TVS., Conclusions: A large multiinstitutional study is indicated to determine if annual TVS screening will cause a significant decrease in site-specific mortality from ovarian cancer.

The growth of a large proportion of estrogen receptor-positive breast tumors is stimulated by estrogen and can often be controlled through antiestrogen therapy. Resistance to antiestrogen (AE) therapy can occur while tumors retain the expression of estrogen receptors (ERc) and remain functionally responsive to estrogens. The ability of specific antiestrogen binding sites (AEBS) to prevent AE from interacting with ERc has been examined as a possible mechanism through which this appropriation of AE could interfere with antiestrogen action. Comparisons were performed between uterine preparations where ERc activity exceeded AEBS binding and liver preparations where AEBS binding predominated. Identical estimates of ERc activity were obtained in uterine preparations using either [3H]estradiol or [3H]-4OH-tamoxifen and radioinert diethylstilbestrol (alpha,alpha'-diethyl-4,4'-stilbenediol) to estimate nonspecific binding. AEBS binding was observed only when [3H]-4OH-tamoxifen was used, while binding to Type II sites was resolved only with [3H]estradiol. When excess AEBS activity predominated, analyses with radiolabeled estrogen and antiestrogen present simultaneously showed that virtually all of the antagonist was bound to AEBS with little of the antagonist available to associate with ERc. In an effort to relate these observations to AE resistance per se, ERc and AEBS were measured in MCF-7 human breast cancer cells (ERc-positive, responsive to estrogens and antiestrogens) and in variant AE-insensitive LY-2 human breast cancer cells (ERc-positive, responsive only to estrogens). In AE-resistant LY-2 cells, the ratio of AEBS:ERc was approximately three times greater than in MCF-7 cells. Examination of 128 human breast carcinomas revealed that AEBS activity was present and could exceed ERc activity. Importantly, the partition of significant AE away from ERc was observed in human specimens. These observations identify a biochemical mechanism for antiestrogen resistance through which AE access to ERc can be totally incapacitated while sensitivity to estrogens continues. These observations indicate that AEBS activity, in addition to ERc activity, may provide helpful information for predicting the response of certain cancers to hormonal therapy.

The influence of estrogen (E) and antiestrogen (AES) on the in vitro growth of BG-1 ovarian carcinoma cells, which express steroid receptors was examined (K. R. Geisinger, T. E. Kute, M. J. Pettenati, C. E. Welander, Y. Dennard, L. A. Collins, and M. E. Berens, Characterization of a human ovarian carcinoma cell line with estrogen and progesterone receptors, Cancer 63, 280-288, 1989). All determinations were simultaneously referenced under similar conditions to MCF-7 cells, a well-established cell line for modeling hormonal responses in breast cancer. In "complete" media containing fetal calf serum (FCS, 10%), MCF-7 cell numbers increased approximately 7 x in 7 days, remaining at this level Days 8-15. In contrast, BG-1 cells achieved similar numbers by Day 7, but showed apparent exponential growth over Days 8-15 to 15-20 x. Phenol red-free media containing 10% FCS (less than 20 pg estradiol (E2)/ml by RIA) was used to assess responses to E and AES. Growth of both MCF-7 and BG-1 cells slowed in E-free media. E2 (10 nM) stimulated the growth of both cell lines, yet was responsible for exponential increases during Days 8-15 only in BG-1 cell numbers (50-70 x). The metabolically active AES (4OH-tamoxifen, 50 nM) reduced E2-stimulated MCF-7 growth to 3-4 x, while tamoxifen (50 nM) had no effect. Rescue with 10 microM E2 fully overcame the AES inhibition of MCF-7 proliferation. In contrast, BG-1 cells experienced significant E2-stimulated growth reductions in the presence of either 4OH-tamoxifen or tamoxifen. E2 was observed to rescue BG-1 cells from both of these antagonists. We conclude that BG-1 ovarian carcinoma cells respond in vitro to E and AES. Moreover, by virtue of responses to tamoxifen, BG-1 cells may have an intrinsic capacity to hydroxylate tamoxifen to its active metabolite. This property of ovarian carcinoma cells might be worth exploiting in the design of more effective combination chemotherapy regimens.

From November 1987 to January 1991, 1300 postmenopausal women underwent screening with transvaginal sonography (TVS). Women eligible for screening were all asymptomatic with no known ovarian tumors. Ovarian volume was calculated using the prolate ellipsoid formula, and a value in excess of 8.0 cm3 was considered abnormal. Ovarian abnormalities were detected in 33 women (2.5%), and 27 underwent exploratory laparotomy. Ovarian tumors were noted in all 27 patients, including 2 primary carcinomas and 14 serous cystadenomas. The two women with ovarian carcinomas had normal results of pelvic examinations and normal serum CA-125 levels. Both women had Stage I disease, and are alive and well after conventional therapy. TVS was time efficient, easy to perform, and well-accepted by patients. Currently, there are more than 3000 patient years of follow-up in the screened population, and there have been no deaths due to ovarian cancer. A multi-institutional trial to determine the efficacy of TVS as a screening method for ovarian cancer is indicated.

16 alpha-[123I]Iodo-17 beta-estradiol (16 alpha-[123I]E2) has been characterized for use as a selective radioligand for estrogen receptor (ERc) that is capable of generating in situ images of ERc-positive tumors. High specific activity 16 alpha-[123I]E2 (7,500-10,000 Ci/mmol) was used in all determinations. Radiochemical purity was determined by thin layer chromatography, and the selectivity of radioligand for ERc was evaluated using size exclusion high performance liquid chromatography on ERc prepared from rodent uteri. Efficiencies of radioidination approaching 100% were achieved, and excellent receptor selectivity was obtained even when the efficiency of radioiodination was as low as 10%. Low radiochemical purity was always associated with poor selectivity for ERc. No new radioligand species was generated during the course of radiodecay; however, reduced binding over time, even when increased activity was used to compensate for radiodecay, indicated that the formation of a radioinert competitor does occur. 16 alpha-[123I]E2 demonstrated stable, high affinity binding to ERc and was concentrated by ERc-positive tissues. After injecting 16 alpha-[123I]E2 in vivo, images of ERc-containing tissues were obtained, including rabbit reproductive tract and dimethylbenzanthracene-induced tumors. The demonstrations of ERc selectivity and image formation both indicate that 16 alpha-[123I]E2 should have promise as a useful new radiopharmaceutical for imaging ERc-positive cancers.

From November 1987 to April 1989, 1000 women 40 years or older underwent screening vaginal sonography at the University of Kentucky Medical Center (Lexington, KY). Patients included in this investigation were all asymptomatic and had no known pelvic abnormalities. Each ovary was measured in three planes and ovarian volume was calculated using the prolate ellipsoid formula. The upper limit of normal for ovarian volume was 18 cm3 in premenopausal women and 8 cm3 in postmenopausal women. In patients with normal scans, mean ovarian volumes decreased from 6.8 cm3 to 3.0 cm3 with menopause. Thirty-one patients (3.1%) had abnormal vaginal sonograms and 24 underwent exploratory laparotomy. All patients undergoing surgery had ovarian or fallopian tube tumors with dimensions identical to those predicted by ultrasound. Histologic diagnoses of these tumors included the following: adenocarcinoma, one, serous cystadenoma, eight; endometrioma, six; and cystic teratomas, two. Vaginal sonography was performed easily and without complications, and was well accepted by patients. All patients with normal sonograms have been rescreened annually and none have subsequently developed ovarian cancer. Further clinical trials to determine the efficacy of vaginal sonography as a screening method for ovarian cancer are indicated.

The efficacy of carbon dioxide laser therapy in the treatment of cervical intraepithelial neoplasia (CIN) was evaluated in 253 patients treated at the University of Kentucky Medical Center from 1984 to 1987. All patients had histologically confirmed CIN and were treated in an outpatient setting. Parameters examined included severity of neoplasia, presence of koilocytosis on biopsy, depth of laser ablation, and the number of cervical quadrants involved by CIN. Following therapy, patients were examined at 3-month intervals from 12 to 48 months (mean 18 months). Eighty-nine per cent of patients were successfully treated with one laser ablation and all patients were cured of disease with two treatments. Over 70% of patients initially failing therapy had CIN III or greater than or equal to 3 quadrant disease. The most significant predictor of failure was lesion size. Only 74% of women with 3 or 4 quadrant disease were successfully treated with one treatment. No patient experienced postoperative infection or bleeding requiring treatment. These data confirm that carbon dioxide laser therapy is a safe and highly effective treatment method for all forms of CIN.

Antagonism to estradiol has been examined in murine uteri. When tamoxifen was administered simultaneously with estradiol (0.05 microgram/mouse), it was able to act as an antagonist over the dosage range 0.05-50 micrograms/mouse. The metabolite 4-hydroxytamoxifen (4OH-tamoxifen) had high affinity for estrogen receptors and was a slightly better antagonist over the dosage range 0.005-1 microgram/mouse. After uteri were exposed to either [3H] estradiol or [3H]4OH-tamoxifen, receptors complexed with [3H] estradiol penetrated a chromatin region, which was released as the Mg2+-soluble chromatin fraction after DNAase I treatment more readily than receptors complexed with [3H]4OH-tamoxifen. [3H]4OH-tamoxifen-receptor complexes could not be driven into the Mg2+-soluble chromatin fraction by increasing the ligand concentration during translocation. Relative to [3H]estradiol, significantly more [3H]4OH-tamoxifen was observed to associate with uterine cells and to penetrate the nucleus so that neither restricted entry nor extranuclear partitioning could explain the failure of [3H]4OH-tamoxifen-receptor complexes to enter the Mg2+-soluble chromatin. Bleomycin, an agent that interrupts DNA continuity, did not interfere with the appearance of estrogen receptor activity in the Mg2+-soluble chromatin fraction. Preincubation of intact uteri in the presence of molybdate (20 mM) did inhibit the appearance of receptor activity in this chromatin fraction; however, this effect did not occur through inhibition of receptor activation, but, rather, through the lowering of receptor activity in all chromatin fractions. In the studies reported here, the chromatin positioning of estrogen receptors complexed with estradiol appeared to be distinct from the positioning of receptors complexed with 4OH-tamoxifen. These observations suggest an additional basis from which the mechanisms separating the actions of estrogen agonists and antagonists can be approached.

The use of VisiCalc and VisiPlot software routines for performing calculations and graphing of data commonly encountered in steroid biochemistry is described. These software routines have utility and are relatively easy to use, requiring little or no programming experience. Use of these routines is demonstrated in terms of data processing associated with chromatography of receptor preparations and equilibrium binding analyses. The execution of these software routines is rapid and can result in a considerable savings of time and personnel. These and other generically similar software routines should have the greatest utility in laboratories where the experimental design is subject to on-going change.

The intranuclear locations at which rat uterine estrogen receptors interact with chromatin have been probed using digestions performed with DNAase I and micrococcal nuclease. Exposure to nuclease has been controlled to effect limited to extensive digestion of nuclear DNA under conditions which maintain the integrity of the [3H] estradiol-receptor complex. The effect of divalent cation concentration on the release of estrogen receptors fron nuclease-treated chromatin was examined and found to be of consequence above 2 mM. Exposure to nuclease released nuclear estrogen receptors from chromatin, with DNAase I being more efficient than micrococcal nuclease in mediating this release. The release of the bulk of nuclear estrogen receptors closely paralleled the nuclease-mediated digestion of chromatin DNA. At 1 h after exposure to estrogen, substantial quantities of uterine estrogen receptors (80-90%) were distributed in chromatin fractions which, on the basis of fractionation terminology, have been termed 'transcriptionally inactive' by convention. Enrichment of estrogen receptors in chromatin which has been termed ' transcriptionally active' only occurred with 10-20% of the estrogen receptors. Hence, our findings support a model where, at early times after estrogen exposure, receptors from the rat uterus are enriched to only a minor extent in chromatin to which 'transcriptional activity' is generally assigned while the bulk of receptors are localized in chromatin which is generally considered 'transcriptionally inactive'.

The physical properties of 59 anticancer agents have been examined with respect to solubility in tissue culture media, binding to ultrafiltration materials, and molecular absorbance and fluorescence behavior. Methods for dissolving these agents, which are compatible with in vitro sensitivity testing of human tumor cells to anticancer agents, are reported in this paper. The potential for anticancer agent binding to cellulose nitrate/cellulose acetate and teflon membrane ultrafilters was documented, and quantitation of these anticancer agents based upon absorbance and fluorescence spectroscopy was performed. Post-filtration quantitation of anticancer agents was found to be a reliable method for determining the actual drug concentrations available in tumor cell sensitivity testing in vitro. The properties documented herein are pharmacologically relevant parameters related to in vitro determinations of human tumor cell sensitivity to anticancer agents.

Surgical specimens from 111 patients with Stage I endometrial cancer were reviewed for the presence of lymph-vascular space invasion by tumor cells. Lymph-vascular space invasion was noted in 16 cases, and occurred most frequently in poorly differentiated tumors with deep myometrial penetration. Tumor recurrence developed in 44% of patients whose tumors demonstrated lymph-vascular space invasion as opposed to only 2% of patients without this finding (p less than 0.001). Of seven patients with lymph-vascular space invasion who experienced tumor recurrence, five developed extra-pelvic metastases. Discriminant function analysis of these data revealed a statistically significant correlation between lymph-vascular space invasion and tumor recurrence, independent of histologic differentiation of myometrial penetration. These findings suggest that lymph-vascular space invasion by tumor cells is an important prognostic variable in Stage I endometrial cancer which should be considered in treatment planning.

After exposing urological tumor cells to anticancer agents in vitro, cellular esterase activity and the ability to exclude propidium iodide (PI) were examined as dual indicators of functionality or "viability." High esterase activity/PI exclusion was observed in assays in which anticancer agents failed to inhibit cellular proliferation, while low esterase activity/PI exclusion was often observed when proliferation had been significantly inhibited. In a number of instances, exposure to anticancer agents did produce significant inhibition of proliferation without lowering viability. In this setting, the recovery of proliferative capacity could be demonstrated with several transitional cell carcinoma cell lines, and this recovery was always associated with high esterase activity/PI exclusion. When the proliferation of primary urological tumor preparations was inhibited by drug exposure, estimates of elevated viability were obtained in 27 per cent of the determinations. Thus, viability estimates may be an indicator of the potential for tumor-cell recovery from exposure to anticancer agents. Moreover, the potential for recovery may explain differences between the results of chemosensitivity testing and actual clinical events by reconciling clinical failures with elevated viabilities indicative of this potential.

The analysis of data generated on a flow cytometer (FCM) is often performed on a computer obtained especially for dedicated use with the flow cytometer. This computer component can be expensive and also presents the FCM user with the added burden of mastering specialized programming language or of accepting the secret analytical processes of protected proprietary program routines. We believe that the evolution of more accurate and efficient FCM analyses that have the power to consider complex signal distributions can be assisted by the availability of analysis programs written in languages common to many users. DNA analysis routines written for a relatively inexpensive microcomputer (IBM PC/XT) in Basic and Pascal are described here. The routines can automatically process multiple FCM data files and can provide high-resolution graphic hardcopy. A foreground/background utilization is also described that allows the computer to be available for other uses in the laboratory.

The estrogenicity of coumestrol, a fluorescent phytoestrogen, has been examined in murine uteri. Coumestrol competed with 17 beta-[3H]estradiol for binding to cytoplasmic estrogen receptors, caused cytoplasmic estrogen receptors to associate with chromatin in the nucleus, and induced progesterone receptors. By use of size-exclusion high-performance liquid chromatography (SEHPLC), the interaction of coumestrol with estrogen receptors was examined directly by monitoring the fluorescence associated with macromolecules having properties characteristic of estrogen receptors. These analyses were made possible by the addition of dimethylformamide to the elution buffer, at a concentration (7.5%) which improved recoveries but did not interfere with estrogen receptor binding. It was possible to detect fluorescent coumestrol at approximately 0.5 nM. All determinations were performed with preparations in which estrogen receptor activity was 3-10 nM. Exposure of these preparations to coumestrol (50 nM) resulted in the elution of increased fluorescent activity in the regions where estrogen receptors eluted during SEHPLC. This fluorescent activity was reduced when diethylstilbestrol, 17 beta-estradiol, hexestrol, or tamoxifen was present as a competitor (2 microM) but was unaffected by testosterone or progesterone. Diethylstilbestrol reduced fluorescence below endogenous base lines and thereby displayed a fluorescence quench property which was not observed with other ligands. When hepatic and renal estrogen receptor preparations were used, the injected receptor activity was observed to be the major limiting factor in detecting the interaction of coumestrol with estrogen receptors. These observations are relevant to attempts to visualize estrogen receptors in tumor cells and demonstrate that accepted biochemical criteria for ligand-receptor interaction can be satisfied when fluorescent ligands are examined.

The polar metabolite of the anti-estrogen alpha-[4-pyrrolidinoethoxy]phenyl-4-methoxy-alpha'-nitrostilbene (CI628), that is selectively accumulated by the nuclear estrogen receptor in vivo, is identified as the corresponding free phenol (alpha-[4-pyrrolidinoethoxyl]phenyl-4-hydroxy-alpha'-nitrostilbene (CI628M) and has been prepared in high specific activity tritium-labeled form to study its interaction with the rat uterine estrogen receptor. CI628M has an affinity for the cytosol receptor that is comparable to that of estradiol (Kd (CI628M) = 0.16 nM; Kd (estradiol) = 0.28 nM), and the binding of these two compounds is mutually competitive. Dissociation of the CI628M complex with the estrogen receptor is somewhat slower than that of the estradiol complex, and prewarming the complexes at 28 degrees C causes a characteristic decrease in the rate of dissociation. On low salt sucrose gradients, the CI628M and estradiol cytoplasmic receptor complexes both sediment as 8S species; their sedimentation profiles on high salt gradients are somewhat different, however. While the estradiol receptor complex shows mainly a 4.6S species that shifts to a 5.8S species upon warming, the unwarmed CI628M complex is predominantly 5.8S and becomes somewhat more disperse upon warming. Under high salt conditions, there is a progressive shift of the 4.6S to the 5.8S species as cytosol concentration increases; the CI628M complex shows a greater tendency to form the more rapidly sedimenting form at all cytosol concentrations. After in vivo injection of [3H]CI628M, the uterine nuclear receptor sediments as a characteristic 5S species. Cytosol receptor complexes with estradiol and CI628M, maintained at 0 degrees C, show very little binding to ATP-Sepharose, while after warming, 50-60% of both complexes are bound by the resin. Both complexes also show similar elution profiles from DNA-cellulose columns; however, the extent of binding of the [3H]CI628M receptor complex to the column is consistently only 40-50% that of the estradiol receptor complex. These results indicate that the properties of the uterine estrogen receptor complexed with estradiol and with the high affinity anti-estrogen CI628M are very similar. The differences observed in the behavior of the CI628M cytosol receptor complex on high salt sucrose gradients and on DNA-cellulose columns, though quantitative rather than qualitative, may be indicative of differences in receptor properties that are important to the antagonist activity of this compound.

Because in vitro cell growth of transitional cell carcinoma explants and cell lines often fail to adequately proliferate in semisolid media, we have examined the effect of agents used to make media semisolid (methyl cellulose, Bacto-agar, Sea Plaque agarose and Sea Prep 15/45 agarose) on the in vitro growth of 11 transitional cell carcinoma cell lines. The growth of human transitional cell carcinoma lines was supported such that agents permissive for growth ranked as follows: Sea Plaque agarose approximately Sea Prep agarose greater than methyl cellulose greater than Bacto-agar. These observations have important implications for the in vitro study of transitional cell carcinoma cell lines and are relevant to the development of improved chemosensitivity determinations for human transitional cell carcinoma.

After exposure to ligand at 0-4 degrees C, estrogen receptors from mouse uteri characteristically eluted between thyroglobulin (Mr 669,000) and ferritin (Mr 443,000) during size-exclusion HPLC. However, when preparations were warmed with ligand under mild activating conditions, most or all of the receptor was observed as a much larger complex, which eluted between dextran blue 2000 and thyroglobulin. Formation of the large complex required ligand, was inhibited by molybdate, and occurred even in 0.4 M KCl. Slower ligand dissociation characterized the large complex, indicating that activated receptors were included preferentially. This large complex did not form when charged cytosols were aged, concentrated, or precipitated, indicating that formation was not the result of random aggregation. After exposure to conditions commonly used for activation (25 degrees C, 60 min), most receptor existed as a very large, monodisperse complex of finite size, predicting an ordered structure for these large complexes that should be useful for defining the types of proteins which can interact with estrogen receptors. Formation of the large complex was not impeded or disrupted by EDTA, RNase, DNase I, thiourea, or mercaptoethanol; however, the capacity to form this large complex was not demonstrated by preparations that had been exposed to trypsin or by the small receptor forms obtained after salt extraction. Proteolytic sensitivity and lack of sensitivity to RNase or DNase indicate that interactions between receptors and other proteins are involved in peak A formation.(ABSTRACT TRUNCATED AT 250 WORDS)

Steroid receptor activity can be more quickly estimated by size-exclusion chromatography than by conventional analysis on sucrose gradients, and profiles of receptor activity are better resolved. Here we discuss several factors affecting this form of analysis in clinical laboratories. The composition of the elution buffer influences steroid receptor elution and recovery: high ionic strength, neutral pH, and the presence of dimethylformamide are important. Of the TSK-SW (Beckman) size-exclusion chromatographic columns we considered, the TSK-G2000SW column appeared to be the most appropriate. "Reference" elution profiles are presented for several marker proteins and estrogen receptor forms generated under different sample-treatment conditions. In examining the sensitivity of receptor analyses by this method, we used fresh rodent preparations, a commercial receptor reference (Estrocept), and human tumor material obtained by needle biopsy. We also compared frozen and lyophilized receptor preparations with fresh ones.

The mouse uterus has been used as a model system with which to examine the interaction of anticancer agents with steroid hormone receptors and to evaluate the effect of a single exposure to a cytotoxic anticancer agent on the subsequent elicitation of the uterotrophic response to estradiol. The uterotrophic response was interpreted in terms of the induction of progesterone receptors, uterine weight gain and increased uterine DNA content. Evaluation of 34 cytotoxic agents selected for this study provided little evidence to substantiate the interaction of these agents with estrogen or progesterone receptors. Although prior treatment with certain cytotoxic agents partially inhibited the subsequent responses to estradiol, some capacity to respond to estradiol was always retained. The majority of cytotoxic agents had little impact on the capacity to respond to estradiol. Thus, in these studies where high sublethal concentrations of cytotoxic agents were administered prior to estradiol, there was no indication that the mechanisms regulating subsequent hormonal responses were compromised.

Immunohistochemical determinations of carcinoembryonic antigen, alpha-fetoprotein, human chorionic gonadotropin (hCG), and human placental lactogen (hPL) were performed on tissue sections from 137 epithelial ovarian cancers. Fewer than 25% of serous cystadenocarcinomas contained detectable amounts of any marker. Carcinoembryonic antigen was present in over 50% of tumors, and was noted most frequently in mucinous, endometrioid, and clear cell carcinomas. hPL was demonstrated in 30% of endometrioid carcinomas but was rarely present in other cell types. Both alpha-fetoprotein and hCG were noted in fewer than 10% of all major cell types of epithelial ovarian cancer. Forty-five patients had serial determinations of plasma levels of carcinoembryonic antigen at the time of regular follow-up visits. Serial plasma levels of carcinoembryonic antigen accurately predicated recurrence in nine of 16 patients whose tumors contained carcinoembryonic antigen, in contrast to two of 16 patients whose tumors were devoid of antigen.

Human tumor cell lines, derived from cancers of the colon, ovary, and cervix, were grown in liquid tissue culture media and media made semisolid with agar (Bacto + deoxycholate lactose agar), agarose [LE, ME, Sea Plaque and Sea Prep (15/45)], and methyl cellulose. The effects of each agent on overall cell proliferation and rate of overall cell proliferation were examined. The agents, used to make media semisolid, were observed to inhibit or, in some cases, enhance cell growth in a fashion that was characteristic of individual cell lines. These phenomena may be of consequence to the optimization of nutrient media for primary tumor cell preparations.

The chemical stability of doxorubicin in a variety of tissue culture media has been studied by thin layer chromatography (TLC). In all the media examined, authentic doxorubicin was converted to a chemically distinct form as evidenced by the failure of this form to migrate on TLC plates. The rates of conversion were rapid enough (t 1/2 approximately equal to 3 hr) to be of consequence in chemosensitivity determinations, especially if working solutions of doxorubicin were to be routinely made and stored in tissue culture media. The addition of certain antioxidants to media did not prevent the conversion of authentic doxorubicin. However, doxorubicin was quite stable in distilled water. No single component of media was found to be responsible for the conversion of authentic doxorubicin, although arginine, histidine, tyrosine, NaHCO3, and Fe(NO3)3 could each generate a form of doxorubicin which did not migrate in TLC analysis. Purification of the nonmigrating form of doxorubicin demonstrated that in vitro conversion resulted in considerable loss of lethality while antiproliferative activity was retained. These observations provide possible explanations for the variability in chemosensitivity determinations and may explain some of the failures to predict clinical responsiveness.

Prognostic parameters were evaluated in 22 patients with small (less than or equal to 2 cm) superficially invasive (less than 5 mm) squamous cell carcinoma of the vulva. Primary surgery included radical vulvectomy with bilateral superficial and deep inguinal lymph node dissection in 11 patients, and wide local excision with ipsilateral superficial inguinal lymph node dissection in 11 patients. Of the 22 patients studied, only 2 (9%) had lymph node metastases. Both patients had a single positive ipsilateral superficial inguinal node. Perineural invasion was strongly associated with lymph node metastases (P less than 0.01). In this group of patients, grade, depth of invasion, lymph-vascular space invasion, and lymphoplasmacytic infiltration were not predictive of lymph node metastases (P greater than 0.05). Two patients initially treated with wide local excision and ipsilateral superficial inguinal lymph node dissection developed recurrent vulvar neoplasia on the contralateral vulva, and both were successfully retreated by wide local excision. All patients are presently alive and well with no evidence of disease. None of the histomorphologic parameters studied were predictive of tumor recurrence. These data suggest that wide local excision with ipsilateral superficial inguinal lymphadenectomy is effective in the treatment of patients with small, superficially invasive carcinomas of the vulva.

A high mol wt estrogen receptor complex (mol wt: greater than 669,000) has been isolated from chromatin prepared from mouse uteri with the use of multiple protease inhibitors, exposure to DNAase I, and size exclusion HPLC (SEHPLC). This complex is stable in the presence of molybdate and 0.4 M KCl. Because receptors in the cytosol do not elute similarly to this large complex when investigated under a wide variety of conditions, it is unlikely that random receptor aggregation is responsible for the formation of this nuclear complex. Consequently, this complex may represent a collection of chromatin components that participate in the mechanisms of hormonal regulation. Since the large nuclear receptor complex can be isolated, this hypothesis can be readily subjected to extensive investigation.

Estrogen receptor activation has been examined in murine uteri by characterizing binding to ATP-Sepharose. Determinations were performed under conditions in which specific binding to estrogen receptors was demonstrated by both agonist and antagonist without participation by nonreceptor antiestrogen-binding components. Cell-free activation of estrogen receptors in cytosol was more effectively promoted by [3H]estradiol estradiol than by [3H]4-hydroxytamoxifen or [3H]tamoxifen aziridine. However, when in situ activation was examined after intact uteri were exposed to [3H]estradiol or [3H]4-hydroxytamoxifen, virtually all extracted nuclear receptors demonstrated activated binding to ATP-Sepharose within 20 min of hormone exposure. Profiles of nuclear receptor activation were remarkably similar after exposure to either agonist or antagonist. Estrogen receptors in cytosol prepared after exposing intact uteri to 3H-labeled ligands were characterized by much less ability to bind to ATP-Sepharose than nuclear receptors. After uteri were exposed to [3H]estradiol, the activated receptor fraction in the cytosol progressively increased in contrast to preparations obtained after uteri were exposed to [3H]4-hydroxytamoxifen, which demonstrated a constant level of activation. Thus, even when activation has occurred within the intact uterus, agonists and antagonists may be characterized by different apparent levels of receptor activation in the cytosol fraction. These differences in the cytosol, however, are considerably overshadowed by the extensive activation occurring within the nuclear fraction, which we have observed to be similar with agonist and antagonist. Since estrogen receptors appear to act within chromatin, and cytosol receptors may be produced by receptor redistribution during preparation, we interpret these observations to indicate that in situ receptor activation is very similar after exposure to either agonist or antagonist. Consequently, antagonism does not appear to be associated with antiestrogens that impede receptor activation within intact murine uteri.

The effects of different agars (Bacto-agar and deoxycholate lactose agar), agaroses (LE, ME, Sea Plaque and Sea Prep 15/45) and methyl cellulose on the growth of a human tumor cell line, derived from a transitional cell carcinoma of the urinary bladder, were examined. The overall growth in the presence of agars and agarose was generally less than in liquid medium alone. In contrast, growth in the presence of methyl cellulose was significantly enhanced. Thus, methyl cellulose may be a useful agent for optimizing the proliferation of primary tissue cultures prepared from human transitional cell carcinomas.

Size-exclusion high-performance liquid chromatography was used to characterize the hydrodynamic molecular properties of estrogen receptors complexed with estradiol and the antiestrogen 4-hydroxytamoxifen. Cytoplasmic estrogen receptors complexed with [3H]-4-hydroxytamoxifen did not undergo reductions in hydrodynamic size after exposure to KCl or urea. Nuclear receptors complexed with 4-hydroxytamoxifen eluted as hydrodynamically larger molecules than nuclear receptors complexed with estradiol. Because identical hydrodynamic characterizations were obtained with the covalent ligand [3H]tamoxifen aziridine, these differences in chromatographic behavior are due to differences in ligand-mediated receptor properties and are not the result of ligand dissociation. When estrogen receptors, complexed with either [3H]estradiol or [3H]-4-hydroxytamoxifen, were exposed to trypsin, the receptors complexed with 4-hydroxytamoxifen eluted as larger hydrodynamic forms than receptors complexed with estradiol. These observations are interpreted to indicate that estradiol and 4-hydroxytamoxifen mediate contrasting transitions in the molecular orientation of estrogen receptors. The consequences of the transitions mediated by 4-hydroxytamoxifen appear to be that intermolecular associations become difficult to disrupt with KCl or urea and that the accessibility of trypsin-sensitive proteolytic sites becomes altered. Chromatin fractionation using DNase I and hypotonic Mg2+ solubilization identified a chromatin region that was less readily penetrated by receptors complexed with 4-hydroxytamoxifen than receptors complexed with estradiol.(ABSTRACT TRUNCATED AT 250 WORDS)

From 1973 to 1987, 16 patients with International Federation of Gynecology and Obstetrics (FIGO) Stage I serous papillary endometrial carcinoma were evaluated and treated at the University of Kentucky Medical Center (Lexington, KY). All patients were 60 years of age or older, and all were postmenopausal. Patients were treated with total abdominal hysterectomy, bilateral salpingo-oophorectomy, and paraaortic lymph node sampling, and 38% were noted to have more extensive disease than appreciated clinically. Nine patients were given adjuvant postoperative radiation. Seven patients (44%) developed recurrent cancer with liver, lung, and upper abdomen being the most common sites of spread. Prognosis was most directly related to the presence of lymph vascular space invasion and the depth of myometrial penetration. No patient with serous papillary carcinoma confined to the endometrium developed recurrent cancer. In contrast, the recurrence rate of patients having myometrial invasion was 70% (P less than 0.03). Hormonal therapy was of limited value in the treatment of recurrent disease. This data suggests the need for adjuvant systemic therapy in the treatment of patients with Stage I serous papillary carcinoma of the endometrium who have myometrial invasion.

Estrogen and progesterone receptors prepared from mouse, rat, and human uteri, as well as from human breast cancers, have been characterized by gel-exclusion high-performance liquid chromatography. The qualitative relationships previously established by sedimentation analysis between the cytoplasmic [aggregated (approximately 8S), deaggregated (approximately 4S), and trypsinized (approximately 3.6S)] and nuclear (approximately 5S) forms of the rat uterine estrogen receptor were maintained by this technique. Differences in the partition of estrogen and progesterone receptors from the same species as well as interspecies differences in these receptors were reproducibly observed. Multiple forms of human estrogen and progesterone receptors could clearly be resolved in a single analysis and were distinct from serum steroid binding tissue contaminants. Separation analyses, performed at flow rates up to 2 mL min-1, were capable of resolving all receptor forms in 10--12 min with the column returning to base line in 25 min. With this exclusion gel column (TSK-G3000SW) as a background upon which to reference different receptor forms, eight distinct partitions or elution positions have been enumerated. This approach has considerable promise for the rapid characterization of different forms of steroid-receptor proteins. Moreover, it should provide a critical advantage in minimizing the opportunities for receptor modification during separation analysis and in maximizing the opportunity to study short-lived interactions between receptors and physiologic or pharmacologic ligands.

From 1962 to 1985, 2201 patients with invasive cervical cancer were staged, evaluated, and treated at the University of Kentucky Medical Center. After a thorough evaluation, 25 cases (1.1%) fulfilled the histologic criteria for small cell cancer defined by Reagan and coworkers. These patients were computer-matched for age, disease stage, and lesion size to 25 patients with large cell nonkeratinizing cancer and 25 patients with keratinizing squamous cell cancer. Morphometric analyses of nuclear size and maximum nuclear diameter were performed on all cases without knowledge of cell type. Small cell cancers were characterized by a nuclear area of 160 mu 2 or less and a maximum nuclear diameter of 16.2 mu, which was significantly lower than that for large cell tumors. Thirty-three percent of the small cell carcinomas stained positively for the neuroendocrine markers (neuron-specific enolase [NSE] and chromogranin [CGR]), whereas the remainder contained only epithelial markers such as cytokeratin (CYK) and epithelial membrane antigen (EMA). Small cell cancers were associated with a high frequency of lymph-vascular space invasion and a diminished lymphoplasmacytic response. Patients with small cell cancer had a significantly higher recurrence rate, particularly to extrapelvic sites, than the matched patients with large cell cancers, and their survival was lower. Clinical trials to determine the efficacy of adjuvant chemotherapy in the treatment of small cell cervical cancer are needed.

The emergence of a near-haploid clone of cells in blast phase chronic myelogenous leukemia is an unusual event. We report such a case and review eight other cases described in the English literature. The significance of the substantial loss of genetic material is discussed as is the phenotypic and genotypic heterogeneity observed in this group of patients.

Tumor markers in gynecologic malignancies can be classified generally as oncofetal proteins, carcino-placental proteins, and more specific tumor-associated antigens. Carcinoembryonic antigen (CEA) is most effective as a tumor marker in mucinous adenocarcinomas of the endocervix and ovary and in keratinizing squamous cell carcinomas of the cervix. In contrast, the use of alphafetoprotein (AFP) in gynecologic cancer is limited to patients with germ cell tumors of the ovary and specifically endodermal sinus tumors. The beta subunit of human chorionic gonadotropin (beta-hCG) remains an exemplary tumor marker for trophoblastic malignancies and may be useful in selected patients with epithelial carcinomas of the ovary. Plasma levels of these antigens are generally related to total tumor burden (tumor antigen concentration x extent of disease)). Although the lack of specificity of these markers has limited their use in the diagnosis of gynecologic malignancies, they have been effective as a means of monitoring disease status in patients whose tumors contain high antigen concentrations. More specific tumor-associated antigens have been described in ovarian cervical cancers, but their clinical efficacy remains to be demonstrated in large numbers of patients. Immunohistochemical staining of tissue specimens identifies patients whose tumors contain high antigen concentrations and who therefore should benefit most from serial plasma determinations following therapy. Potential future uses of biochemical markers include radioimmunodetection procedures using radiolabelled antibodies to tumor-associated antigens and antigen-directed chemotherapy.