Pathological review of breast lesions analyzed for estrogen receptor protein.

This report provides a detailed pathological review of 333 specimens analyzed for estrogen receptor protein (ERP) and correlates a series of morphological features with ERP results. Included were 147 primary breast carcinomas, 78 metastases, 27 fibroadenomas, and 81 nonneoplastic tissues, all from women. ERP in cytosols was assayed by incubation with [3H]estradiol in the presence and absence of "cold" estradiol followed by dextran-charcoal treatment. Results were summarized as positive (greater 60% inhibition by nontritiated estradiol, greater than 10 fmoles/mg protein), negative (less than 60% inhibition by nontritiated estradiol, less than 10 fmoles/mg protein), or intermediate borderline combinations. ERP in primary tumors ranged from 0.2 to 358 fmoles/mg protein (54.4% positive, 35.4% negative, 10.2% borderline). New findings are: (a) a high frequency of positive ERP in invasive lobular carcinoma (12 of 13, 92.3%) compared to typical ductal tumors (64 of 117, 54.7%); and (b) low frequency of positive ERP(5 of 21, 23.8%) in tumors with a prominent local lymphocyte reaction. Three ERP-positive noncarcinomatous specimens were fibroadenomas of high epithelial cellularity from patients under 30 years. No statistically significant relationship existed between ERP and any other morphological features that were examined.

9-Cis retinoic acid inhibits growth of breast cancer cells and down-regulates estrogen receptor RNA and protein.

All-trans retinoic acid (tRA) inhibits growth of estrogen receptor-positive (ER+) breast cancer cells in vitro, and a variety of retinoids inhibit development of breast cancer in animal models. 9-cis retinoic acid (9-cis RA) is a naturally occurring high affinity ligand for the retinoid X receptors, as well as the retinoic acid receptors (RARs). Whether 9-cis RA has a different spectrum of biological activity from tRA, which only binds RARs with high affinity, is largely unknown. We studied the effects of 9-cis RA on growth and gene expression in ER+ and ER- human breast cancer cells. 9-cis RA inhibited the growth in monolayer culture of several ER+, but not ER-, cell lines in a dose-dependent manner. Growth inhibition and morphological changes by 9-cis RA were similar to those of tRA, suggesting that the ability to bind both RAR and retinoid X receptors did not significantly augment growth inhibition or confer sensitivity to tRA-resistant lines. MCF-7 cells exposed to 9-cis RA showed a dose-dependent accumulation in G1. Northern analyses showed that RAR-alpha and RAR-beta were not significantly regulated, while RAR-gamma was up-regulated and retinoid X receptor alpha was down-regulated by 9-cis RA. Since interactions between tRA and ER-dependent transcription have recently been reported, we investigated whether these retinoids regulate expression of ER itself or estrogen-responsive genes. Both 9-cis RA and tRA induce down-regulation of ER mRNA and protein in MCF-7 cells. 9-cis RA down-regulates expression of the estrogen-responsive genes PR and pS2 in MCF-7 cells as reported previously for tRA. In several ER-positive subclones, we found that the degree of ER expression and regulation, but not always estrogen-sensitivity, correlates with the growth-inhibitory effects of 9-cis RA. Further, in an ER-, retinoid-unresponsive breast cancer cell line, induced ER expression confers responsiveness to retinoid growth inhibition. These data, combined with reports of additive growth inhibition of tRA and tamoxifen in vitro, suggest that 9-cis RA might augment the ability of tamoxifen to inhibit growth of ER+ breast cancer cells in vivo.

Phase I trial with pharmacokinetic analyses of high-dose carboplatin, etoposide, and cyclophosphamide with autologous bone marrow transplantation in patients with refractory germ cell tumors.

Thirty patients with cisplatin-refractory germ cell tumor were treated with high-dose carboplatin, etoposide, and cyclophosphamide and autologous bone marrow transplantation. The total dose of carboplatin was 1500 mg/m2, etoposide 1200 mg/m2, and cyclophosphamide was increased by increments from 60 to 150 mg/kg. Twenty-five cycles of treatment, given to 17 patients, did not include granulocyte-colony stimulating factor (G-CSF). Nineteen cycles of high-dose chemotherapy, given to 13 patients at the 2 highest dose levels of cyclophosphamide, included G-CSF. The dose of cyclophosphamide was escalated to 150 mg/kg/cycle without prohibitive toxicity. The use of G-CSF resulted in a shorter duration of neutropenia (P = 0.07); the median number of days until the recovery of an absolute granulocyte count > 0.5 was 25 without G-CSF and 14 with G-CSF. The most frequent nonhematological toxicity was hepatic, and there were 2 (7%) treatment-related deaths. Thirteen (43%) patients achieved a complete response, and 8 are alive and free of disease (27%); 7 are in continuous complete response (23%), and 1 after resection of a solitary site of disease following a relapse after high-dose chemotherapy. Five patients had pharmacology studies performed to determine the area under the curve (AUC) of free and total platinum, carboplatin, etoposide, cyclophosphamide, and phosphoramide mustard. There was a decrease in the AUC of cyclophosphamide and an increase in the AUC of phosphoramide mustard, the "active" metabolite, with successive days of treatment. The interpatient variability of the AUC of cyclophosphamide/phosphoramide mustard that was demonstrated was most likely a result of each individual's metabolic capacity. The measured AUC of carboplatin and/or free platinum closely approximated the predicted AUC of carboplatin calculated by renal function in 3 of the 5 patients. In summary, cyclophosphamide administered at a dose of 50 mg/kg x 3 days was achieved with acceptable toxicity, and no further dose escalation is planned. High-dose carboplatin, etoposide, and cyclophosphamide achieved a 23% continuous complete response proportion (27% alive, free of disease) when used as third-line therapy in germ cell tumor patients refractory to cisplatin + ifosfamide-based chemotherapy. Ongoing studies are addressing the role of high-dose carboplatin-containing chemotherapy in previously untreated patients with poor prognostic features or as a part of first-line salvage.

Intrapleural cisplatin and mitomycin for malignant mesothelioma following pleurectomy: pharmacokinetic studies.

PURPOSE: Intrapleural cisplatin-based chemotherapy has been used in the treatment of patients with malignant pleural mesothelioma and malignant pleural effusions, but the pharmacokinetics of this form of chemotherapy have not been previously evaluated. We performed pharmacokinetic studies on 12 patients who received both intrapleural cisplatin and mitomycin immediately following pleurectomy/decortication for malignant pleural mesothelioma. PATIENTS AND METHODS: Simultaneous pleural fluid and plasma samples were collected at 15 and 30 minutes, and at 1, 2, 3, 4, and 24 hours after administration of the intrapleural chemotherapy (cisplatin 100 mg/m2 and mitomycin 8 mg/m2), and after cisplatin (total and free) and mitomycin levels were measured. The mean peak levels, the areas under the concentration-time curve (AUC) and the drug half-lives (t1/2s) in plasma and pleural fluid were compared using the paired t test. Differences were considered significant if P less than or equal to .05. RESULTS: Systemic absorption was rapid, with peak plasma levels being reached within 1 hour of administration of the intrapleural chemotherapy. Peak plasma levels measured after intrapleural chemotherapy approximated those reportedly attained during systemic administration of these drugs at similar doses. However, the mean peak cisplatin and mitomycin levels, and their mean AUCs, were significantly higher in the pleural fluid than in the plasma. There was a three- to fivefold advantage (on a logarithmic scale) for pleural to plasma AUCs for both cisplatin and mitomycin. The mean t1/2s for cisplatin and mitomycin were significantly longer in the plasma than in the pleural fluid. CONCLUSIONS: The pharmacokinetics of intrapleural cisplatin-based chemotherapy are analogous to those of intraperitoneal chemotherapy. Our findings show that intrapleural cisplatin-based chemotherapy has a distinct local pharmacologic advantage, but also produces significant and sustained drug plasma levels.

Phase I trial of high-dose tamoxifen as a modulator of drug resistance in combination with daunorubicin in patients with relapsed or refractory acute leukemia.

Tamoxifen and its main metabolite N-desmethyltamoxifen (NDMTmx) have been shown to increase intracellular daunorubicin (DNR) levels in human leukemia cell lines that display the multidrug resistant (MDR) phenotype. We designed a phase I dose escalation study of Tmx (200-700 mg/day p.o. for 7 days) in combination with a fixed dose of DNR (50 mg/m2 intravenously on days 5, 6 and 7) in patients with advanced leukemia to determine whether this combination could be given safely and whether plasma levels of 10 microM, the effective in vitro MDR modulator concentration, could be achieved. Pharmacologic studies of Tmx, NDMTmx and DNR, and its main metabolite daunorubicin-ol (DNR-ol) were performed as was determination of P-glycoprotein (Pgp) using a monoclonal antibody that recognizes an external epitope of the molecule. A total of 14 patients (median age 50, range 22-67) were treated at the following dose levels: 200 mg/day: three patients; 400 mg/day: four patients; 550 mg/day: three patients; and 700 mg/day: four patients. Two patients with relapsed AML achieved remission. Toxicity of the combination was similar to that seen with DNR alone and no severe hepatic, cardiac or retinal toxicity was noted. Plasma Tmx levels approached 7 microM at the two highest dose levels studied; plasma levels of NDMTmx were slightly less. The area under the curve for DNR and its main metabolite daunorubicin-ol (DNR-ol) did not show significant changes with escalation of Tmx dose. This phase I study suggests that concentrations of Tmx high enough to reverse the MDR phenotype can be approached and that the combination of high-dose Tmx with a standard dose of DNR has an acceptable toxicity profile. More evaluation in phase II studies is necessary to define further its role as an MDR modulator.

Measurement of the estradiol receptor in human breast tissue by the immobilized antibody method.

A simple immobilized antibody procedure for estradiol receptor detection has been applied to samples of human breast tissue cytosols previously assayed by the charcoal-dextran method. Good agreement between the two procedures was realized confirming the validity of the immobilized antibody assay.

The acute monocytic leukemias: multidisciplinary studies in 45 patients.

The clinical and laboratory features of 37 patients with variants of acute monocytic leukemia are described. Three of these 37 patients who had extensive extramedullary leukemic tissue infiltration are examples of true histiocytic "lymphomas." Three additional patients with undifferentiated leukemias, one patient with refractory anemia with excess of blasts, one patient with chronic myelomonocytic leukemia, one patient with B-lymphocyte diffuse "histiocytic" lymphoma and one patient with "null" cell, terminal deoxynucleotidyl transferase-positive lymphoblastic lymphoma had bone marrow cells with monocytic features. Another patient had dual populations of lymphoid and monocytoid leukemic cells. The true monocytic leukemias, acute monocytic leukemia (AMOL) and acute myelomonocytic leukemia (AMMOL), are closely related to acute myelocytic leukemia (AML) morphologically and by their response to chemotherapy. like AML, the leukemic cells from the AMMOL and AMOL patients form leukemic clusters in semisolid media. Cytochemical staining of leukemic cells for nonspecific esterases, presence of Fc receptor on the cell surface, phagocytic ability, low TdT activity, presence of surface "ruffles" and "ridges" on scanning EM, elevations of serum lysozyme, and clinical manifestations of leukemic tissue infiltration are features which accompanied monocytic differentiation in these cases.

Estrogen and progesterone receptor content in human thyroid disease.

Recent studies have confirmed the presence of estrogen and progesterone receptors in many benign and malignant thyroid tumors, but their clinical significance is unclear. The estrogen and progesterone receptor content of 135 thyroid lesions was assayed prospectively from 1980 through 1986 using the dextran-coated charcoal method. The cases included 30 papillary, 13 follicular, 6 medullary, and 2 Hürthle cell carcinomas. Thirty-two follicular adenomas, 45 goiters, and 7 cases of thyroiditis also were studied. Estrogen receptor protein was positive (> or = 2 fmol/mg) in 46% of the cases, with no clear statistical predilection related to the type or size of the thyroid lesion, age, or sex. Progesterone receptor protein was positive (> or = 10 fmol/mg) in 51% of the cases, with the highest median values obtained in papillary carcinomas, particularly in male patients and women older than 50 years. Metastases at presentation, noted in 28 of 51 carcinomas, were unrelated to receptor content. Mean follow-up of 55 months in 48 patients with various carcinomas yielded 12 cases with late metastases, which were similarly unrelated to receptor content. Although estrogen receptors are commonly detectable in thyroid lesions, they have no clear relationship to presenting clinical or pathologic features or, in cases of carcinoma, to subsequent metastatic potential. The role of progesterone activity in papillary carcinoma and in goiter merits more investigation.

Estrogen receptor protein of breast cancer in patients with positive nodes. High recurrence rates in the postmenopausal estrogen receptor-negative group.

In 448 patients with positive axillary lymph nodes who were treated with mastectomy at Memorial Sloan-Kettering Cancer Center, New York, from 1973 to 1978, estrogen receptor (ER) status was associated with survival. With a median follow-up of 75 months, significant differences were noted in the 288 postmenopausal patients; ER-positive patients had better six-year disease-free survival than ER-negative patients (60% vs 45%), as well as better overall survival. These differences were true in subgroups with one to three and four or more involved nodes. The addition of adjuvant systemic therapy had no significant effect on either ER-positive or ER-negative patients. The need for new imaginative systemic programs in the subgroup of ER-negative postmenopausal patients with breast cancer with positive nodes is apparent.

Carboplatin-based chemotherapy with pharmacokinetic analysis for patients with hemodialysis-dependent renal insufficiency.

Three patients with renal insufficiency requiring hemodialysis were treated with carboplatin at 100 mg/m2 in combination with etoposide for advanced germ-cell tumor (GCT, two cases) or Adriamycin + vinblastine for a transitional-cell carcinoma of the ureter (one case). Hemodialysis was performed 24 h after the administration of carboplatin. Both patients with GCT achieved a complete response, and the patient with transitional-cell carcinoma of the ureter was inevaluable for response but his disease has not progressed. The dose of carboplatin was increased in one patient as renal function improved on therapy. In two patients, the pharmacokinetics of carboplatin were determined; the pre-dialysis half-lives, AUC, and total body clearances of free carboplatin-derived platinum were estimated to be 32 and 18.3 h, 4.93 and 6.17 mg ml-1 min, and 18.2 and 18.7 ml/min, respectively. The dialysis elimination half-lives (t1/2 beta) of 2 and 3 h, respectively, for these two patients were markedly lower than the predialysis values, indicating that carboplatin was dialyzed. In summary, carboplatin can be given to patients with severe renal insufficiency. Adequate AUCs were achieved and dialysis limited systemic exposure to free carboplatin.

Estrogen receptor protein in visceral metastases from breast carcinoma.

Estrogen receptor protein assays were done on 1,243 tissue specimens, 34 of which represented metastases from primary breast carcinoma to parenchyma of liver, lung or brain. Eight of these specimens contained significant amounts of estrogen receptor protein. Most (4 of 5 evaluable cases) of the ERP-positive patients responded to endocrine ablation, as compared with 13 percent (2 of 16 evaluable cases) of the ERP-negative patients. We conclude that biopsy of liver and lung metastases may be useful in some circumstances and that endocrine manipulation frequently provides palliation when these metastases are ERP-positive.

Performance evaluation of a blood gas system in a central and satellite laboratory.

In many hospitals blood gases are performed in several laboratories. It is important to establish that values obtained in all laboratories are identical and that the same quality control program is used. Evaluation of the performance of blood gas apparatus has been made in two of our laboratories. Result reproducibility was assessed by means of gas analyzer system control. Measurements were made in duplicate of patients' specimens in the same instrument and duplicate measurements of patient samples with both instruments. A13 day evaluation using this system was carried out in both laboratories during all shifts and showed excellent agreement with the manufacturer's reported values. The combined standard deviation for pH at all levels was 0.009,pCO2 -0.83 mmHg and pO2 - 3.9 mmHg. Patients' samples analyzed in the same instrument showed an absolute average difference between duplicates for pH of 0.003, pCO2 - 0.62 mmHg and pO2 -0.87 mmHg. The comparison of duplicate samples in the two instruments in the different laboratories showed an absolute average difference for pH of 0.009,pCO2-1.7mmHg and pO2 - 1.6 mmHg.

Barr body distribution and estrogen receptor protein in mammary carcinoma.

The study was undertaken to investigate the relationship between Barr body distribution and estrogen receptor protein content of mammary carcinoma. The proportion of cells with one or more Barr body was determined in 105 specimens of mammary carcinoma from Guard stained imprints. Receptor protein content of the specimen was measured by the dextran charcoal method and compared with histopathologic features of the carcinomas. Primary carcinomas with Barr bodies in more than 10 percent of tumor cells were more likely to have higher levels of receptor protein than those with a lower proportion of Barr body containing cells (P less than 0.005). The results obtained for primary carcinoma may explain why patients with carcinomas that have a high proportion of Barr body positive cells are more likely to respond to hormonal therapy. Furthermore, these observations, when correlated with other available data about ERP suggest that an X-chromosome is involved in the synthesis of and/or carries the locus of action for estrogen receptor protein.

Estrogen binding and estrogen receptor activity in the human prostate: a preliminary report.

Assay of estrogen receptor activity in prostates from patients who ranged in age from 22 to 78 years and had not received any previous hormonal therapy was carried out by incubation of cytosols with (3)H-estradiol in the presence and absence of excess, nonradioactive estradiol. Hyperplastic prostatic tissues were used in the study. The kinetics of each reaction were studied and analysis of the data revealed 3.4 to 35.7 femtomoles of receptor protein per mg of cytosol protein; the dissociation constants obtained from a Scatchard plot ranged from 1.1 × 10(-10) to 1.2 × 10(-8)M.The small number of patients prevents realistic quantitative assessment of the apparent estrogen binding activity demonstrated in these preliminary studies, but the qualitative identification of such activity provides possible grounds for further insight into the hormonal mechanisms in the pathophysiology of prostatic diseases and of their responses to endocrine therapy.

Chemical and physical properties of the disulfides of bovine neurophysin-II.

Bovine neurophysin-II is shown to be very susceptible to partial reduction in the absence of urea. Reduction of an average of one disulfide leads to major changes in conformation and disulfide optical activity, manifest in part by pronounced far-uv ellipticity changes, complete loss of the 248-nm ellipticity band, and a shift of the 278-nm ellipticity band to shorter wavelengths with loss of half its intensity; the reduction process generates a mixture of products and appears to be accompanied by disulfide interchange. The circular dichroism data indicate that the disulfide(s) most susceptible to reduction or interchange are either the principal contributors to the 248- and 278-nm ellipticity bands or that the optical activity of other disulfides is dependent on their integrity. Peptides that bind to the hormone-binding site of neurophysin-II protect against reduction. On reoxidation of partially reduced neurophysin-II there is only a partial return of the native circular dichroism spectrum and electrophoretic behavior. The percentage of native protein in samples reoxidized following different degrees of reduction was estimated by comparison of the circular dichroism spectra of these samples with those of the fractionated native and denatured components of monoreduced-reoxidized neurophysin. Under our reoxidation conditions, less than 50% native protein was found in monoreduced-reoxidized neurophysin and less than 10% native protein was found in completely reduced-reoxidized neurophysin. The results are interpreted with qualified reference to a model in which one or more disulfides are "strained" in the native state and in which the native protein is unstable relative to species in which the disulfides are differently paired.

Estrogen receptor protein of breast cancer as a predictor of recurrence.

To assess the value of estrogen receptor protein (ERP) as a predictor of tumor recurrence, 556 patients treated by mastectomy between 1973 and 1978 for primary operable breast cancer had ERP determination of their tumors. All patients had histologically negative nodes. Two hundred fifty-six patients were ERP-positive, 233 were ERP-negative, and 67 were ERP-borderline. ERP-borderline patients showed recurrence and survival figures similar to ERP-negative patients and were grouped with them in the analysis. With a median follow-up of 75 months, overall survival for the entire group at 72 months was 93% with a disease-free survival of 85%. No difference in either overall survival or DFS was noted between the ERP-positive (94% and 83%, respectively) and ERP-negative (91% and 86%, respectively) groups. The incidence of recurrence in premenopausal women was 12% (14/115) in the ERP-negative patients versus 17% (9/48) among the ERP-positive patients. This contradicts the current impression that ERP-negative, premenopausal patients have a poorer prognosis than ERP-positive patients. Postmenopausal patients had a 16% recurrence in both ERP-negative (27/171) and ERP-positive (31/202) categories. On the basis of 6-year median follow-up, it was concluded that ERP status is not an indicator of recurrence. In particular, patients with negative nodes should not be considered for adjuvant chemotherapy on the basis of negative estrogen receptor status of their primary tumors.

Levels of estrogen and progesterone receptor proteins in patients with breast cancer during various phases of the menses.

Analysis of estrogen and progesterone receptor proteins was carried out in 75 premenopausal and 79 postmenopausal patients with primary operable breast carcinoma who were treated from January 1983 to December 1984. The frequency of estrogen receptor protein positive/progesterone receptor protein positive (+/+); estrogen receptor protein negative/progesterone receptor protein negative (-/-); estrogen receptor protein negative/progesterone receptor protein positive (-/+); and estrogen receptor protein positive/progesterone receptor protein negative (+/-) was 40.5%, 30.5%, 23%, and 6% in premenopausal patients, respectively, and 52%, 24%, 2.5%, and 21.5% in postmenopausal patients, respectively (p less than 0.001). The mean positive estrogen receptor protein concentration (expressed as femtomoles per milligram of protein +/- SEM) was significantly higher in postmenopausal patients (54 +/- 6) than in premenopausal patients (19 +/- 2) (p less than 0.005). The progesterone receptor protein values did not differ significantly between these two groups. The phase of the menstrual cycle was recorded at the time of surgery in the 75 premenopausal women. Maximum receptor positivity occurred in the secretory phase, however, this difference is not statistically significant, and our data suggest that there are no distributional differences between the phase of menses and positivity of estrogen and progesterone receptor proteins. Future studies which included analyses of circulating sex steroid levels and receptor proteins will provide a better understanding of complex hormonal regulatory mechanisms which exist in patients with breast cancer.

Pilot study of 5-azacytidine (5-AZA) and carboplatin (CBDCA) in patients with relapsed/refractory leukemia.

5-azacytidine (5-AZA) and carboplatin (CBDCA) are two agents which have demonstrated antileukemic activity in a number of phase I-II trials. Their mechanisms of action and pharmacology related to cell resistance suggested suitability for combination therapy. The aim of this pilot was to evaluate the effects of this combination in the treatment of patients with relapsed/refractory acute leukemia. A total of 21 patients was enrolled. 5-azacytidine, at doses ranging from 50-150 mg/m2/day, was administered as a 2-hr infusion for 5 consecutive days. On day 3, patients began a 5-day course of CBDCA given as a 24-hr continuous intravenous infusion of 250 mg/m2/day. There were no complete remissions with this regimen. Although there were three partial responses, these were generally of short duration. Nonhematologic toxicities were mild. No correlation was seen between response and serum platinum levels. These results demonstrate that the 5-AZA/CBDCA combination is ineffective therapy for heavily pretreated patients with acute leukemia.

A preliminary evaluation of tissue polypeptide antigen in serum or urine (or both) of patients with cancer or benign neoplasms.

We evaluated assays of Tissue Polypeptide Antigen in serum and urine, as an index to the presence of cancer. In the assay, serum, which is first absorbed with human albumin-labeled sheep erythrocytes, or untreated urine (diluted with an equal volume of TPA-free serum) is incubated with antibody specific to Tissue Polypeptide Antigen and then reacted with sheep erythrocytes labeled with Tissue Polypeptide Antigen. We found an increased concentration of Tissue Polypeptide Antigen in the serum of 378 of 513 (74%) and in the urine of 49 of 77 (64%) patients with cancer, as compared with 40/112 (36%) and 7/29 (24%), respectively, for individuals with benign neoplasms. Normal individuals were defined as those with less than 0.09 unit of the antigen per milliliter of specimen. Concentrations exceeding this were found in 2/67 (3%) sera and 6/56 (11%) urines from supposedly normal persons. Tissue Polypeptide Antigen was found in above-normal concentrations in patients with cancer, regardless of neoplasm type and extension, with a higher proportion of abnormal values in patients with distal metastases.