Estrogen and progesterone receptor and c-erbB-2 oncoprotein analysis in pure in situ breast carcinoma: an immunohistochemical study.

The authors studied 30 consecutive archival cases of pure breast carcinoma in situ (CIS) for estrogen receptor (ER), progesterone receptor (PR), and c-erbB-2 oncogene product. Clinical factors of age and menstrual status and histologic features of tumor type and nuclear grade were determined. Some 77% represented ductal CIS, with 10% noninvasive papillary, and 13% lobular CIS. A total of 22 of 30 (73%) were ER+ and 19 (63%) were PR+; 24 (80%) were c-erbB-2+ with 11 (37%) showing strong staining; 75% of the ER-PR group had tumors of NG 3, versus 14% for the ER+PR+ plus ER+PR- groups. Nonstatistically significant trends correlating the strongly positive oncogene group with PM status, receptor negativity, and high nuclear grade were noted. The overall rate of receptor positivity, as well as the correlation with nuclear grade, is similar to that in invasive carcinomas c-erbB-2 protein expression shows a higher rate in in situ than is reported for invasive tumors, and shows a tendency to be expressed more strongly in tumors with other features of poor prognosis. Therefore, these factors may also have prognostic significance in CIS. Continued followup of this population to recurrence may provide further insight.

Pathologic changes associated with androgen deprivation therapy for prostate cancer.

Prostate glands exposed to androgen deprivation with leuprolide +/- flutamide were evaluated for pathologic changes which might be related to therapy. Comparing pretreatment and posttreatment tissue by visual discrimination using light microscopic study revealed treatment-related alterations in the size and distribution of neoplastic glands in 60% of cases. Quantitative measurements documented glandular changes in an even greater percentage of cases. Although distinctive, the histologic pattern was not specific for leuprolide/flutamide. The absence of appreciable degeneration and necrosis in tumor cells suggests that this type of androgen deprivation may act through suppression rather than ablation of prostatic cancers. The relationship between treatment-related histologic effects and initial tumor grade and clinical stage as well as expression of prostate-specific antigen was studied. Accurate histologic assessment of leuprolide/flutamide-treated prostate glands should not be a problem so long as specimens are thoroughly examined and drug-related variations in tumor morphologic features are appreciated.

Pulse labeling of hydroxyproline and leucine in reproductive tissues: a model for assessment of degree of reactivity.

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.

Application of DNA probes to hematology: an overview with selected examples.

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.

Carboxyhemoglobin levels in patients with flu-like symptoms.

Subacute carbon monoxide poisoning is commonly misdiagnosed as an influenza-like viral illness. All patients presenting to the triage nurse at University Hospital with flu-like symptoms during February 1985 were asked to give blood samples for carboxyhemoglobin determination. Fifty-five patients (10% of those eligible) with headache, dizziness, nausea, vomiting, diarrhea, weakness, general malaise, or shortness of breath were enrolled in the study. Carboxyhemoglobin levels ranged from 0 to 21%. Thirteen patients (23.6%) of this self-selected subgroup had carboxyhemoglobin levels greater than or equal to 10%. There was no statistically significant difference in carboxyhemoglobin levels between smokers and nonsmokers. More patients using wood heat had elevated carboxyhemoglobin levels than patients using any other form of heating (P less than .05). No patient with a carboxyhemoglobin level greater than or equal to 10% was diagnosed as having subacute CO poisoning by emergency physicians. Physicians must seek out the possibility of CO toxicity in patients with flu-like illness, particularly in inner-city populations during the heating months. Fundoscopy and COHb levels may be useful in selected cases to correctly diagnose patients and avoid a return to a hazardous environment with potentially fatal consequences.

Steroid related neoplasia in human liver.

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.

Fine-needle aspiration of breast cancer. Relationship of clinical factors to cytology results in 689 primary malignancies.

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.

Comparison of corticosteroid therapy in the prevention of pelvic tissue reaction and adhesion formation.

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.

The study of fluorescent probes by quantitative video intensification microscopy (QVIM).

In this paper we describe a system for the quantitation and display of fluorescence at the cellular level. It uses a low light level video camera which is interfaced to a fluorescence microscope and to a microprocessor-controlled video digitizing system. With the use of a light pen entry system one can specify areas of the field for measurement. The data obtainable are the area and perimeter of the delimited zone, the distribution of pixel intensities within this zone over a 16-level gray scale, and a value for total fluorescence intensity. Statistical outputs for repeated measurements are also obtained. The system responds linearly to light input, has a high degree of reproducibility, and provides good spatial resolution. Using the DNA-specific dye, Hoechst 33248, in diploid fibroblasts as test material, the system is shown to be able to reproduce expected distributions for amounts of DNA per cell. The capabilities and advantages of pseudocolor display are also demonstrated. We conclude that, in conjunction with appropriate fluorescent probes, systems such as the one described make it possible to do quantitative histochemistry of living cells and to measure substances not previously amenable to study.

Cytochrome P-450 activity in human leiomyoma and normal myometrium.

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.

Evaluation of danazol influence upon the uterus using scanning electron microscopic morphometric and biochemical analyses.

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.

Studies on the role of Ca++ in cell division with the use of fluorescent probes and quantitative video intensification microscopy.

It is clear that QVIM systems, when combined with appropriate fluorescent probes can be utilized to perform quantitative cytochemical studies on living and fixed cells. They also have the potential to facilitate studies of substances which like Ca++ are not easily studied by other means. The preliminary studies we have described support the idea that calcium ions and calcium transport enzymes may indeed play important roles in cell division and indicate that the tools we have at hand should help us further our understanding of the mitotic process.

Epidermal growth factor binding by breast tumor biopsies and relationship to estrogen receptor and progestin receptor levels.

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.

Measurement of fluorescence using digital integration of video images.

The authors describe a system for the quantitation of fluorescence light output by individual cells using the signal obtained from a silicon intensifier target video camera. The video image is digitized to 4 bits (16 levels), and a 512 X 512 matrix is constructed and stored in 128K of video memory. Areas to be measured are user-specified by means of a light pen entry system. Recorded intensities are integrated under microprocessor control to provide a measure of total fluorescence in the selected areas. The distribution of light intensities per pixel over the 16-level gray scale as well as morphometric data are also obtainable. Linear response to transmission and fluorescence standards was verified. Reproducibility of the system was evaluated using fluorescent beads and glutaraldehyde-fixed chick red blood cells, which gave coefficients of variation comparable to those obtainable from other systems. Measurements of DNA per nucleus of human diploid fibroblasts using Hoechst dye 33258 yielded the two sharp peaks corresponding to the 2C and 4C values of DNA expected from such cells. We conclude that digital video measurement of fluorescence provides meaningful data and has considerable promise as a sensitive tool for the quantitation of fluorescence at the cellular level.

Estrogen receptor analysis by flow cytometry.

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.

Binding sites for epidermal growth factor in human uterine tissues and leiomyomas.

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.

Prostaglandin E and F2 alpha receptors in human uterine leiomyomas.

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.

Topography of human uterine prostaglandin E and F2 alpha receptors and their profiles during pathological states.

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).