A prospective study evaluating tissue reactivity by light microscopic and pulse labeling-autoradiographic techniques is presented. One uterine horn of a Sprague-Dawley rat model was subjected to transection and reanastomosis using microsurgical techniques (control). The opposite horn of each was also cross-sectioned with the application of an absorbable topical hemostatic agent (Avitene) and then reanastomosed. After closure of the abdomen, the animals were randomly assigned to groups according to the planned day of sacrifice (days 3, 7, 14, 21, or 28). Autoradiographic techniques utilizing [3H]hydroxyproline or [3H]leucine as well as light microscopy were employed to determine tissue reactivity. The results demonstrated an increase in stromal (P less than or equal to .05) and smooth muscle (P less than or equal to .01) reactivity with [3H]leucine labeling, and an increase in smooth muscle (P less than or equal to 0.05) reactivity with [3H]hydroxyproline in the Avitene-treated groups. It is concluded that pulse labeling with tritiated leucine and hydroxyproline is an effective method of objective analysis of tissue reactivity in reproductive organs.

Recent advances in molecular genetics have allowed development of short segments of desoxyribonucleic acid (DNA) which are complementary to active genetic sites. By pairing normal DNA with these "molecular probes", the presence of specific genetic sequences may be detected in patient samples. The majority of hematologic diseases arise from hereditary errors, neoplastic change or parasitic organisms. These types of disorders are particularly suited to diagnosis and management using detection of abnormal DNA sequences. Sensitive laboratory techniques employing these molecular probes are now available directly to detect target DNA sequences, assess abnormal short sequences of DNA, and evaluate the presence of abnormal DNA from its transcription products. Using DNA polymerase amplification, the sensitivity of these methods approaches its theoretical limitation of a single gene pair. In hematology, a prevalent application has been the detection of clonal molecular rearrangements which serves as a marker for lymphocytic neoplasia. Other applications of these techniques include the detection of abnormal chromosomal regions such as the Philadelphia chromosome and the detection of deletions of DNA which occur in such diseases as the myeloid dysplastic syndrome. As more human genetic sequences are known, it seems likely that these techniques will become prevalent among methods of laboratory testing.

Prior to the early 1970's, benign liver neoplasms were among the rarest of tumors. The seemingly rapid increase, especially in young females ingesting oral contraceptives, as well as the catastrophic presentation of many of the tumors resulting from liver rupture and hemoperitoneum, stimulated studies by several investigators. In the Liver Tumor Registry at the University of Louisville, we have examined the histologic material, and finalized the data on 227 tumors, the majority in young women. With few exceptions, they had used oral contraceptives or were either pregnant or immediately post-partum and presumably in a hyperestrogenic state. There have been 82 hepatocellular adenomas (HCA), 105 cases of focal nodular hyperplasia (FNH), and 31 hepatocellular carcinomas. The hepatocellular carcinomas occurred in non-cirrhotic livers, and 14 of the 31 cases were of a distinct, but rare type, polygonal cell carcinoma with lamellar fibrosis. While it seems reasonable to believe steroids play a role in adenomas and in FNH it is less certain that they produce hepatocellular carcinomas since malignant liver tumors are not uncommon in this age group without the use of oral contraceptives. With an estimated 50 million women either currently using or who have used oral contraceptives the risk must be very slight.

Between 1980 and 1983, 689 women with primary breast cancer at the Royal Infirmary of Edinburgh and associated Hospitals had fine-needle aspiration biopsies prior to definitive surgery. Clinical factors relating to the success of these aspirations were evaluated. The most significant factor was which physician performed the aspiration. Size of the lesion was also an important variable; however, size difference could not account for the marked variation between different individuals performing the aspiration. There was no difference between node-positive and node-negative patients when matched by tumor size. A significantly lower rate of positive aspirations occurred in the 45 to 55 year age group which could not be accounted for by tumor size. Location of the mass was not significant, although there was a persistent lower rate of positive aspirates from the upper inner quadrants. Aspiration cytology was positive or suspicious in 65 percent of patients with primary breast cancer who had clinical diagnoses of benign breast lesions. It is concluded that the most significant variable in the accuracy of breast aspiration biopsy is the size of the lesion and the proficiency of the individual performing the procedure. With a skilled physician, positive aspiration results were obtained in over 80% of breast cancers.

The purpose of this research was to compare the effect of glucocorticoids administered in equivalent doses with respect to inflammatory response, fibrosis and adhesion formation. The animal model used was the Sprague-Dawley rat. The surgical technique involved incision and eversion of a portion of the proximal uterine horn. The glucocorticoids administered included methylprednisolone acetate (Medrol, The Upjohn Company), 6.25 mg/kg; hydrocortisone acetate (Hydrocortisone, Merck Sharp and Dohme), 25 mg/kg; betamethasone phosphate (Celestone Phosphate Injection, Schering Corp.), 1 mg/kg; and dexamethasone sodium phosphate (Hexadrol, Organon Pharmaceuticals), 1 mg/kg. These glucocorticoids were compared with a control solution of normal sodium chloride for injection (Abbott Laboratories), 0.5 mL/rat. Four weeks after the initial surgery, the uterine horns were histologically evaluated for inflammatory response, fibrosis and adhesion formation. Betamethasone phosphate produced a significant reduction (P less than 0.05) in fibrosis when compared with all other corticosteroid or control solutions. There was no statistically significant difference in the degree of inflammation or adhesion formation.

Variations in cytochrome P-450 levels may influence the responsiveness of uterine and breast tissue as well as carcinomas to endocrine therapy and may be of particular importance with agents such as tamoxifen (Nolvadex) where hydroxylation is known to alter therapeutic activities. Therefore, a sensitive spectrophotometric assay of cytochrome P-450 levels in reproductive tissue microsomes was developed to measure cyclohexane hydroxylase activity. Cyclohexane served as a substrate for several forms of cytochrome P-450. Human uterine leiomyomas (uterine fibroid tumor) contained significantly higher (p less than 0.01) cytochrome P-450 activity than adjacent normal myometrium. Specific activities for both leiomyomas (2.87 +/- 0.26 nmol/min/mg) and normal myometrium (1.60 +/- 0.11 nmol/min/mg) were in the range of those observed for untreated rabbit liver microsomes (1 to 3 nmol/min/mg). The contribution of smooth muscle in the specimen, the phase of the menstrual cycle, and the clinical diagnosis did not influence the level of cytochrome P-450 activity.

A study using a Sprague-Dawley rat model was designed to correlate morphologic and intracellular steroid receptor alteration with danazol therapy. One uterine horn was removed for cytoplasmic estrogen receptor analysis following four to eight days of danazol therapy. This was followed by intra-aortic injection of buffered phosphate solution and 3 per cent glutaraldehyde. The remaining uterine horn was removed for scanning electron microscopic and morphometric analysis of light microscopic changes. After four days of treatment, there was an increase in the nuclear to cytoplasmic ratio compared with that of the control tissue. Vaginal smears on the eighth day of danazol exposure indicated that the rats had entered a noncycling estrus state, and there was a significant increase in the estrogen-specific binding capacity of the endometrial cells. Scanning electron microscopy showed a distinct decrease in endometrial folds, microvilli and glandular openings, which suggests a decline in secretory activity in the danazol-treated group. Thus, danazol induced measurable changes in the estrogen binding capacity of endometrial cells, and these changes correlated with certain morphologic alterations in the endometrial lining.

A fluorescently labeled estradiol, N'-fluoresceino-N'-(17 beta-estradiol hemisuccinamide) thiourea (FE) was used for measuring estrogen receptor content per cell in tumor cells. The cellular content of FE was measured quantitatively by flow cytometry. Binding of FE occurs in the nanomolar concentration range, an indication of the high affinity of the labeled estradiol. Competition of FE for binding sites is observed with estrogens, but not with progestins, androgens, or glucocorticosteroids, indicating the specificity of FE binding. In contrast to other estrogen receptor assays, this new technique requires a small sample size (about 5000 cells) and permits the assessment of heterogeneity in estrogen receptor expression among tumor cells.

There is no published data regarding whether human uterine tissues and leiomyomas contain binding sites for epidermal growth factor (EGF). The present study was undertaken with 33 myometria, 13 leiomyomas, and 4 endometria. All myometria (fundus) and leiomyomas and 3 of 4 endometria specifically bound 125I-labeled mouse EGF (myometria: mean, 1.1; range, 0.1-3.9 fmol/mg protein; leiomyomas: mean, 1.1; range, 0.2-2.6 fmol/mg protein; endometria: mean, 1.0; range, 0.0-3.1 fmol/mg protein). [125I]EGF binding to myometrium was ligand specific in that only unlabeled EGF, but not unlabeled insulin, hCG, human PRL, prostaglandin E1, or prostaglandin F2 alpha, competed with [125I]EGF for binding. The apparent dissociation constants and specific binding capacities for myometrium and leiomyoma were: 0.7 nM, 1.9 fmol/mg protein; and 0.1 and 3.7 nM, 0.1 and 4.4 fmol/mg protein, respectively. While smooth muscle content decreased from the fundus to the cervical end of uteri, [125I]EGF binding did not correspondingly decrease (r = 0.5; n = 4). The binding of [125I]EGF to myometrium did not vary with the phase of the menstrual cycle or the patients' diagnosis before hysterectomy (P greater than 0.1). In summary, these results demonstrate that human uteri and leiomyomas contain specific, high affinity binding sites for EGF, but the binding neither exhibited topographical changes nor varied with the phase of the menstrual cycle or benign pathological state of the tissue.

9uman uterine leiomyomas specifically bound less (P less than 0.01) [3H]prostaglandin E1 ([3H]PGE1) and [3H] PGF2 alpha than adjacent normal myometria [leiomyomas: mean [3H]PGE1, 16.4 (range, 11.1-25.2) fmol/mg protein; mean [3H]PGF2 alpha, 4.7 (range, 0.8-12.1) fmol/mg protein; adjacent normal myometria: mean [3H]PGE1, 41.7 (range 27.1-60.7) fmol/mg protein; mean [3H]PGF2 alpha, 7.8 (range, 4.3-16.3) fmol/mg protein]. The lower binding of both [3H]PGs by leiomyomas was due to lower numbers of available high and low affinity sites. Leiomyomas and normal adjacent myometria bound 4-7 times more [3H]PGE1 than [3H]PGF2 alpha, and this appears to be due to high affinity and high numbers of low affinity PGE sites. The smooth muscle content was lower (P less than 0.01) in leiomyomas (mean, 28.0%; range, 9.7-45.5%) than that of adjacent normal myometria (mean, 58.9; range, 51.4-71.2%). In summary, this is the first demonstration of PGE and PGF2 alpha receptors in human uterine leiomyomas. Lower receptor numbers in leiomyomas appear to be due to the lower smooth muscle content of the tissue.

Epidermal growth factor (EGF) may be important in regulating the growth of some breast cancer cells in vivo because of its mitogenic action on some breast cancer cell lines in vitro. Epidermal growth factor receptors (EGF-R) were measured in a series of breast tumors to determine what percentage of breast tumors express EGF-R and whether EGF-R was independent of expression of estrogen receptor and progestin receptor. Specific binding of 125I-EGF to membranes from pooled homogenates of breast tumors reached equilibrium after 45 min at 25 degrees and remained constant. Scatchard analysis of 125I-EGF binding indicated a single class of receptors with an apparent Kd of 2 nM and a binding capacity of 28 fmol/mg of membrane protein, and the binding of 125I-EGF was not effectively competed for by insulin, fibroblast growth factor, growth hormone, or prolactin. Specific binding of 125I-EGF of 1 fmol or greater/mg of membrane protein and 15% or greater specific binding was detected in 48% of 137 unselected primary and metastatic breast tumors. The frequency distribution of EGF binding values was unimodal, with a progressive decrease in the proportion of patients with high EGF binding values. The values of EGF binding ranged from 1 to 121 fmol/mg of protein, with an arithmetic mean of 8.4 fmol/mg of protein and a geometric mean of 3.2 fmol/mg of protein. Forty-two % of 24 metastatic breast tumors were positive for EGF binding, with an arithmetic mean of 6.3 fmol/mg of protein and a geometric mean of 4.1 fmol/mg of protein. The magnitude of EGF binding in individual tumors was independent of either estrogen receptor or progestin receptor levels, although the highest quantities of EGF binding were expressed by tumors lacking steroid receptors. Approximately 20% of the tumors in the study were EGF-R-positive and ER-negative, suggesting that the growth of these tumors may be regulated predominantly by a peptide hormone (EGF) rather than a steroid hormone (estrogen). EGF binding did not correlate significantly with age of the patients. Correlation analysis between EGF binding and the percentage of malignant and nonmalignant cell types present in sections of tumor adjacent to the area assayed for EGF binding indicated that the percentage of malignant cells is an important factor in determining the amount of EGF binding in tumor homogenates. The recent discovery of transforming growth factors which interact with the EGF-receptor system suggests additional roles for EGF receptors in breast cancer.

The topography of prostaglandin (PG) E and F2 alpha receptors in uteri of premenopausal women was investigated by dividing uteri into six equal longitudinal strips and further dividing each strip into approximately 1-cm segments. Tissue for determination of smooth muscle content using the Trichrome stain was taken from each section, and the remainder was homogenized for binding studies with 3H-labeled PGs. The [3H] PGE1 binding (mean, 41.5 fmol/mg protein; range, 23.1-58.3) was about 8-fold greater in the fundus than [3H]PGF2 alpha binding (mean, 4.8 fmol/mg protein; range, 1.3-13.0), and this trend was found in most uterine sections. The binding of both 3H-labeled PGs decreased from fundus to cervix, and this decrease was similar to the decrease in smooth muscle content. Scatchard analysis revealed apparent dissociation constants (Kds) of 1.4 and 76 nM and apparent specific binding capacities (Ns) of 25 and 488 fmol/mg protein for [3H]PGE2, and Kd values of 11.5 and 81 nM and Ns values of 19.4 and 58 fmol/mg protein for [3H]PGF2 alpha in the uterine fundus. The Kd values for [3H]PGE2 were similar in other sections of the uterus, but the Ns values were smaller in the lower uterine body and cervical end. While the phase of the menstrual cycle did not influence [3H]PG binding, the diagnosis of abnormal uterine bleeding compared to dysmenorrhea was associated with an increase in [3H]PGE1 binding (P less than 0.05).

Since 1973 a number of investigators have reported an association between liver neoplasia and steroid usage. Through referral material we have examined the histology of over 250 cases of hepatic neoplasia, most in patients receiving steroid medications. The majority have been benign, predominantly focal nodular hyperplasia (55%) and hepatocellular adenoma (39%). The average age was 31.4 years; 83% had significant steroid exposure with an average duration of 71 months for focal nodular hyperplasia and 79.6 months for hepatocellular adenoma. The type of estrogenic agent was predominantly mestranol; however, during the period mestranol was the most frequently used synthetic steroid. A distinct clinical entity of life threatening hemorrhage from the lesion occurred in 31% of patients with hepatocellular adenoma and 9% of patients with focal nodular hyperplasia. Recurrence of benign tumors has occurred in some patients who continued using steroids and regression has been observed in patients who had incomplete tumor removal but discontinued steroid medication. Medial and intimal vascular changes have been present in a large number of the benign tumors. The relationship of these vascular changes to oncogenesis is unclear, but similar lesions have been described in the peripheral vasculature associated with steroid administration. A number of hepatocellular carcinomas have also been seen. Of significance is the young age of these patients and lack of abnormal histology in adjacent nonneoplastic liver. A striking number of the malignant hepatocellular tumors have been of the uncommon type described as "eosinophilic hepatocellular carcinoma with lamellar fibrosis." The epidemiology of liver lesions within this series is difficult to assess, since the material has been referred from very diverse locations.

This study examines experience with over 250 liver tumors in young women. Most were oral contraceptive related. There were two distinct benign liver tumor types: focal nodular hyperplasia and liver cell adenoma. Six benign liver tumors were examined for estrogen receptors. They did not contain significant quantities of estrogen receptor, supporting experimental studies of an epigenetic origin. Multiple tumors occurred in about 20% of cases but did not change the favorable prognosis associated with benign tumors. The most significant source of morbidity and mortality was spontaneous hemorrhage or rupture. Twenty-three women in this series had hepatocellular carcinoma and the majority of these were associated with prolonged steroid usage. These malignant liver tumors occurred in young females without cirrhosis or other factors known to be associated with hepatic malignancy. Unlike “hepatocellular carcinomas” reported in males taking anabolic androgenic steroids, the tumors in females had a high rate of metastasis to a variety of organs. The risk of liver tumors in oral contraceptive users remains unknown, but must be very small since an estimated 150 million women worldwide and 40 million in the U.S.A. have used oral contraceptives.