Rat mammary gland differentiation in vitro in the absence of steroids.

Mammary glands from several strains of immature female rats will form small lobules of alveoli when cultured in chemically defined media lacking steroid hormones but containing insulin and prolactin. The degree of lobulo-alveolar differentiation is increased if estradiol, progesterone, and aldosterone are also included in the culture media.

In vitro stimulation of human breast tissue by human prolactin.

Normal human breast tissue was cultured with defined media plus hormones. The epithelium survived at least 4 days in culture but did not grow in the absence of hormones. Both insulin and human prolactin stimulated growth, but ovine prolactin did not.

Breast gross cystic disease fluid analysis. I. Isolation and radioimmunoassay for a major component protein.

Human breast gross cystic disease (GCD) fluid was analyzed by sodium dodecyl sulfate-acrylamide gel electrophoresis, and four major proteins (GCDFP-70), GCDFP-44, GCDFP-24, and GCDFP-15) were identified. By fractionation techniques, these proteins were separated from one another. The GCDFP-70 was immunologically identical to human albumin and was present in GCD fluid at approximately a 100-fold lower concentration than in plasma. The GCDFP-44 was immunologically identical to human plasma Zn-alpha2-glycoprotein; however, it was present in GCD fluid at an approximately 50-fold higher concentration than in plasma. The GCDFP-24 was the major component protein of GCD fluid. It had progesterone binding activity, and immunologically it was identical to a component of human plasma; however, antisera that identified 30 separate components of plasma failed to identify the GCDFP-24 as one of these plasma proteins. The GCDFP-24 concentration in GCD fluid was approximately 100-fold higher than the plasma analog. The GCDFP-15 component was immunologically distinct from any plasma components, as judged by Ouchterlony analysis. It was, however, immunologically identical with a component of both human milk and saliva. As revealed by radioimmunoassay, plasma levels in normal subjects were 7-85 ng/ml. In patients with metastatic breast carcinoma, markedly plasma levels (150-30,000 ng/ml) of this protein were detected. Short-term tissue cultures of breast carcinoma explants released this protein into the culture medium.

Androgen stimulation of gross cystic disease fluid protein and carcinoembryonic antigen in patients with metastatic breast carcinoma.

Plasma levels of carcinoembryonic antigen (CEA) and the 15,000 molecular weight gross cystic disease fluid protein (GCDFP-15) were determined in 30 patients with metastatic breast carcinoma before, during, and after treatment with fluoxymesterone. Within 2 weeks after initiation of treatment, plasma levels of GCDFP-15 increased 50% above basal values in 15 (79%) of 19 patients. Similar increases in plasma CEA levels occurred in only 5 (23%) of 22 patients. Eight (33%) of 24 patients achieved increases in GCDFP-15 of 500% or more above basal levels after 14-336 days of therapy. Within 2 weeks of fluoxymesterone termination, 14 (93%) of 15 patients had a decrease in plasma GCDFP-15 levels, and in 12 (80%) the decrease exceeded 33% (the inverse of a 50% increase). Conversly, only 5 (33%) of 15 patients experienced a decrease in plasma CEA levels within 2 weeks of therapy termination, and in only 1 (6.7%) subject did the decrement exceed 33%. Nine (90%) of 10 patients who had 50% increases in plasma GCDFP-15 during initial androgen therapy also had significant decreases in plasma GCDFP-15 following termination of therapy. Data on 3 prospectively studied patients demonstrated that plasma GCDFP-15 rose within 24 hours of initiation of fluoxymesterone therapy and continued to rise for at least 6 days. Increased plasma levels of GCDFP-15 were reflected in increased urinary excretion of the glycoprotein.

Carcinoembryonic antigen in nonhuman primates.

Blood levels of carcinoembryonic antigen (CEA) have been measured in several nonhuman primate species. Only gorillas and chimpanzees were found to have significant elevations of CEA-like activity in their blood compared to the normal values of less than 2.5 ng/ml in humans. The average CEA level in 134 chimpanzees was 25.2 ng/ml (range, 4.2--95 ng/ml) and in 13 gorillas it was 32 ng/ml (range, 12.4--61.9 ng/ml). These levels were not related to sex. Blood levels repeatedly taken over a 1 1/2-year period remained relatively stable in both species. Analysis of parallelism of immunologic reactivity showed chimpanzee CEA to be similar to but not identical with human CEA. The molecular size of chimpanzee CEA was also similar to that of human CEA.

Stimulation of insulin secretion by a rapid intravenous calcium infusion in patients with beta-cell neoplasms of the pancreas.

The effects of calcium on fasting plasma insulin and glucose levels were compared in 16 normal subjects and 11 patients with beta-cell neoplasms of the pancreas. Calcium was administered iv either as a rapid calcium infusion (RCI; 2 mg/kg in 1 min) or as a long calcium infusion (LCI; 12 mg/kg in 3 h). In normal subjects, the RCI produced a rise in mean plasma insulin from 11 +/- 1 (+/- SEM) microU/ml basally to a peak of 18 +/- 2 microU/ml (P less than 0.001). No consistent pattern of change in insulin levels occurred during the LCI, and plasma glucose levels did not change significantly with either test. In the patients with beta-cell neoplasms, the RCI resulted in a rapid increase in mean plasma insulin from 36 +/- 6 microU/ml to a peak level of 312 +/- 67 microU/ml (P less than 0.002). With the LCI, a more gradual rise in insulin from 35 +/- 11 to 92 +/- 36 microU/ml occurred (P less than 0.002). The mean increase in insulin in the patients with beta-cell neoplasms was significantly greater for the RCI than for the LCI (P less than 0.01). Pronounced increments in plasma insulin occurred in all 11 patients after the RCI, but in only 3 of 8 patients during the LCI. Plasma glucose levels declined significantly from 69 +/- 7 to 56 +/- 8 mg/dl during the RCI (P less than 0.05) and from 69 +/- 8 to 49 +/- 7 mg/dl during the LCI (P less than 0.005). Symptomatic hypoglycemia developed in 3 patients during the LCI but did not occur after the RCI. These data indicate that calcium is a more effective insulin secretagogue in patients with beta-cell neoplasms when administered as an RCI than as an LCI, and suggest that the RCI may be a useful test for the diagnosis of insulin-secreting tumors.

Progression of postoperative residual medullary thyroid carcinoma as monitored by plasma calcitonin levels.

BACKGROUND: Patients operated on for medullary thyroid carcinoma (MTC) frequently have persistent elevated plasma calcitonin concentrations after operation, indicating remaining tumor. The plasma calcitonin concentration in a patient with MTC roughly reflects the endogenous tumor burden. The only effective treatment for MTC is surgical. The decision about whether a patient with persistent MTC should have a repeat operation would be influenced by knowledge of the natural course of the disease. METHODS: Forty patients with persistently elevated peak plasma calcitonin concentrations after thyroidectomy for MTC were monitored for a mean of 6 years. Serial determinations of plasma calcitonin levels were obtained before and after intravenous injection of calcium and pentagastrin. RESULTS: At the first postoperative test 63% of the patients had undetectable basal calcitonin values, although their stimulated plasma calcitonin concentrations were elevated. The mean annual increase in stimulated plasma calcitonin concentrations was 117%, but plasma calcitonin concentrations were stable in three patients and decreased in one patient. Five patients are known to have experienced distant metastases. CONCLUSIONS: MTC is a progressive disease in most patients with persistent hypercalcitoninemia after thyroidectomy. Stimulated peak plasma calcitonin levels are more meaningful than basal levels in the serial postoperative evaluation of patients with persistent hypercalcitoninemia after thyroidectomy for MTC.

Reoperation for recurrent or persistent medullary thyroid cancer.

BACKGROUND: Initial operations for medullary thyroid cancer (MTC) frequently do not eradicate all disease, as evidenced by persistently elevated levels of stimulated plasma calcitonin. METHODS: Thirty-two patients with MTC and elevated stimulated plasma calcitonin levels after thyroidectomy were studied between 1990 and 1993. Thirty-five repeat neck explorations and microdissections were performed. Four patients also underwent a median sternotomy and mediastinal dissection. RESULTS: In nine patients (group 1), stimulated plasma calcitonin levels were undetectable after reoperation, whereas in 13 cases (group 2) the calcitonin levels decreased by 40% or more. In 10 cases (group 3) the CT levels did not decrease. Primary tumors that exhibited invasive features (invasion of adjacent structures or extranodal or extracapsular spread) were found more often in patients from group 3 than in patients from groups 1 or 2 (p < 0.05, Fisher's exact test). CONCLUSIONS: Reoperation resulted in normalization of calcitonin levels in 28% of patients and a decrease in calcitonin levels by 40% or more in another 42% of patients. The data also suggest that patients whose primary MTC has invaded tissues beyond the thyroid gland or a lymph node capsule are less likely to benefit from repeat operation.

Presymptomatic identification of carriers of the multiple endocrine neoplasia type 2A gene using flanking DNA markers.

BACKGROUND: Because the predisposition locus for multiple endocrine neoplasia type 2A (MEN2A) has been mapped to chromosome 10 by genetic linkage analysis, it has become possible to identify gene carriers by following the transmission of linked genetic markers from affected parents to offspring at risk for MEN2A. We have applied a highly accurate genetic test to presymptomatic diagnosis of gene carriers in several large kindreds with MEN2A. METHODS: DNA was extracted from 300 individuals in six kindreds with MEN2A and used for genotyping studies with DNA markers flanking the MEN2A locus. Genotype data were used to predict the inheritance of the MEN2A gene in kindred members at risk according to previously calculated map distances and the program LINKAGE: RESULTS: Ninety-five percent of individuals were informative with markers flanking the MEN2A locus. Of 130 patients at risk, 26 (20%) were predicted to be MEN2A gene carriers, 100% (77%) were noncarriers, and 4 (3%) were recombinant and their gene carrier status could not be determined. Gene carrier prediction probabilities were calculated at greater than 98% in 94% of these patients. CONCLUSIONS: We conclude that genetic testing with flanking DNA markers is a highly accurate method for the presymptomatic identification of MEN2A gene carriers and allows for diagnosis at an earlier stage than does traditional calcitonin testing.

Biochemical correlates of morphologic differentiation in human breast cancer.

In 115 breast carcinoma tissues, histologica grade and cell cytosol concentrations of estrogen receptor (ER) and progesterone receptor (PR) and two breast cyst fluid proteins (gross cystic disease fluid protein [GCDFP-15] and nonreceptor progesterone-binding protein [PBP]) were deterMined. Higher levels (expressed as femtomoles per milligram of protein) of ER (128 +/- 28 versus 11 +/- 1, P less than 0.001) and PR (82 +/- 16 versus 3 +/- 1, P less than 0.001) were found in grade 1 (well-differentiated) carcinomas as compared with grade 3 (poorly differentiated) carcinomas. Similarly, higher concentrations (expressed as nanograms per milligram of cytosol protein) of GCDFP-15 (2110 +/- 840 versus 210 +/- 40, p less than 0.001) and PBP (4920 +/- 1200 versus 370 +/- 60, P less than 0.001) were found in grade 1 as compared with grade 3 carcinomas. Tumor cytosols that contained low levels of both cyst proteins (less than 225 ng/mg GCDFP-15 and less than 750 ng/mg PBP) had a high incidence of grade 3 (35 of 46, 78%) or grade 2 (15 of 46, 33%) histologic findings and had a high incidence of receptor-negative specimens (27 of 52, 52%). Based on these cutoff levels, grade 2 lesions were subdivided into a "high" cyst protein group, which had ER and PR levels similar to grade 1 tumors (93.1 +/- 26.7 for ER and 84.7 +/- 32.4 for PR, P greater than 0.3), and a "low" group, which had receptor values similar to grade 3 carcinomas (14.1 +/- 5.3 for ER and 9.1 +/- 5.2 for PR, P less than 0.3). Although the mean cytosol content of carcinoembryonic antigen (CEA) was significantly higher in malignant tissues (125 +/- 27 ng/mg cytosol protein) than in benign tissues (4.8 +/- 1 ng/mg cytosol protein), the CEA content was not significantly different between grades 1 and 3 tumors.

Hormone response of benign hyperplastic prostate tissue in organ culture.

Glandular benign prostatic hyperplasia specimens from 10 patients were culutured for 4 days in media containing either insulin alone, insulin plus estradiol, insulin plus testosterone, insulin plus human placental lactogen, insulin plus human placental lactogen plus testosterone, or insulin plus human growth hormone plus testosterone. Growth of the specimens was defined by pulsing 3H-thymidine on day 3, terminating on day 4, and determination of acid-insoluble radioactivity by scintillation counting. The response of each specimen was related to its base line response to insulin media. Six of nine specimens were stimulated 80 per cent or more by insulin plus placental lactogen plus testosterone, but only one of seven was stimulated by insulin plus testosterone. Four of 10 were stimulated by insulin plus estradiol. Histologic and autoradiographic results indicated that all growth occurred in the epithelium.

Improved results of cervical reoperation for medullary thyroid carcinoma.

OBJECTIVE: The purpose of the study is to determine whether reoperation for medullary thyroid carcinoma (MTC), performed with low morbidity in carefully selected patients, consistently results in improvement as determined by lowering of stimulated calcitonin levels. BACKGROUND: Persistent or recurrent elevation of stimulated plasma calcitonin levels occurs in > 50% of patients after primary operation for MTC. Success of reoperation with clearance of metastatic cervical nodal disease has been hampered by failure to identify patients with distant metastases and by inadequate removal of involved nodal groups. METHODS: Since 1992, the authors have evaluated 115 patients with recurrent or residual MTC. Fifty-three patients have not undergone operation because of extent of disease, previous extensive treatment, medical condition, or patient choice. Sixty-two patients underwent surgery. Ten patients had laparoscopic or open examination of the liver, the results of which showed liver metastases. Seven patients had palliative debulking of cervical tumor. In 45 patients without evidence of distant metastases, cervical operation was carried out with curative intent. Removal of central, upper mediastinal, and lateral nodes (levels II, III, IV, VI, and VII) was done. RESULTS: Seven of eight patients who had palliative resections are alive without symptoms. In patients who underwent curative resections, postoperative stimulated calcitonin levels were in the normal range in 17 patients (38%) and were not significantly lowered in 6 patients (13%). There were no deaths, and no transfusions were used. CONCLUSIONS: These results are a significant improvement over the authors' previous series and reflect better preoperative identification of patients with disease confined to the neck and improved operative strategy based on knowledge of the pattern of nodal spread of MTC.

Secretion of breast gross cystic disease fluid proteins by T47D breast cancer cells in culture--modulation by steroid hormones.

The effect of steroid hormones on modulating the secretion rates of three human breast gross cystic disease fluid proteins (GCDFP-15, GCDFP-24, and GCDFP-44) by T47D breast carcinoma cells in tissue culture was evaluated. Androgens (dihydrotestosterone or fluoxymesterone) were capable of stimulating the secretion rates for all three GCDFP's while showing a minimal trend toward slowing the growth rate of T47D cells. This is the first study which shows that androgens can specifically stimulate all three of the major breast GCDFP's concomitantly. Progesterone, and three synthetic progestins, all showed inhibition of the growth rate of T47D cells while causing enhancement of the secretion of GCDFP-15 and GCDFP-44, and only minimal effect on the secretion rate of GCDFP-24. Estradiol was essentially neutral to the growth rate of the T47D cells in our test system. Estradiol did cause a mild enhancement of GCDFP-44 secretion rate, with no appreciable effect on GCDFP-15 or GCDFP-24 secretion rates. These findings suggest that an androgenic stimulus may be involved in the secretion of GCDFP's associated with breast gross cystic disease.

Hyperparathyroidism associated with the enlargement of two or three parathyroid glands.

Eighty-five (23%) of 375 patients undergoing surgery for primary hyperparathyroidism were found to have enlargement (greater than 50 mg) of two or three parathyroid glands. Of 76 patients followed from 12 to 140 months after surgery, eight (10.5%) developed hypercalcemia at 1, 4, 45, 64, 74, 79, 84, and 133 months. In a comparison of pertinent preoperative biochemical and pathologic data between 55 patients with two- or three-gland hyperparathyroidism and 55 age- and sex-matched patients with single-gland hyperparathyroidism, only the preoperative serum phosphate differed significantly, being lower in the patients with single-gland disease (2.4 +/- 0.1 vs. 2.6 +/- 0.1; p less than 0.04). In the eight patients with two- or three-gland hyperparathyroidism who developed postoperative hypercalcemia, the preoperative concentrations of serum calcium were lower (10.8 +/- 0.2 vs. 11.5 +/- 0.2; p less than 0.019), the preoperative concentrations of serum phosphate were higher (3.1 +/- 0.2 vs. 2.5 +/- 0.1; p less than 0.020), and the weights of the excised parathyroid tissues were less (356 +/- 72 mg vs. 1354 +/- 215 mg; p less than 0.02) than those of patients with two- or three-gland disease who did not develop postoperative hypercalcemia, indicating a milder form of hyperparathyroidism. In the 68 patients without recurrent hypercalcemia, there was no tendency for the serum calcium concentration to increase with time. Patients with primary hyperparathyroidism associated with two or three enlarged parathyroid glands have an appreciable incidence of persistent or recurrent hypercalcemia, which may increase even further with longer observation.

Immunologic and steroid binding properties of the GCDFP-24 protein isolated from human breast gross cystic disease fluid.

A major protein of human breast cyst fluid, termed GCDFP-24, has the property of specifically binding progestins. The purified glycoprotein, of 24,000 apparent molecular weight, bound pregnenolone and progesterone with highest affinity. The association constant for binding of progesterone was 1 X 10(6)L/mol by Scatchard analysis, and there was one binding site per molecule. Changes to the progesterone structure at C-17, C-20, or C-21 interfered with binding. The pH optimum for binding was 4-4.5. The purified protein was highly stable and was not irreversibly denatured by 50% methanol or 3M guanidine. However, dithiothreitol reversibly interfered with progesterone binding. Rabbit antiserum produced against the glycoprotein recognized an immunologically identical component in normal human sera, and partially cross-reacting components in normal monkey and baboon sera. The component in human sera was present in Cohn fractions IV and VI.

Long-term evaluation of patients with primary parathyroid hyperplasia managed by total parathyroidectomy and heterotopic autotransplantation.

Since 1973, we have performed total parathyroidectomy and forearm parathyroid autotransplantation in 36 patients with generalized (four gland) primary parathyroid hyperplasia. Twenty (56%) patients had nonfamilial parathyroid hyperplasia (NFPH) and 16 (44%) patients had familial parathyroid hyperplasia (FPH). Twenty-one patients (Group A) were undergoing operation for the first time and 15 (Group B) were having either second, third or fourth re-explorations for persistent hyperparathyroidism. All patients in Group A and nine patients in Group B had parathyroid resection and immediate autotransplantation as a single procedure. Six Group B patients had hyperfunctioning parathyroid tissue resected, cryopreserved, and subsequently grafted when it was evident that they had been rendered aparathyroid. A sustained differential elevation (13.7 fold +/- 2.7) of parathyroid hormone was detected in the antecubital vein of the grafted compared to the nongrafted arm in 35 (97%) patients. Two (5.6%) of the 36 patients (both with FPH; one Group A and one Group B) required permanent oral calcium and vitamin D replacement therapy and one (3%) patient (NFPH: Group A) had persistent hypercalcemia postoperatively, presumably due to a supernumerary gland. The remaining 33 (92%) patients became normocalcemia after surgery and 23 (70%) of them remained so. Ten (30%) of the 33 patients developed recurrent graft dependent hyperparathyroidism. Eight patients were from the group with FPH (8/14, 57%) and two were from the group with NFPH (2/19, 11%)(FPH vs. NFPH, p < 0.005). Because of symptoms of hypercalcemia or a serum calcium concentration exceeding 11 mg/dl, partial graft resection was performed in five patients and four became normocalcemic. Patients with generalized primary parathyroid hyperplasia may be difficult to cure, especially if the disease is familial. The technique of total parathyroidectomy and heterotopic autotransplantation of fresh or cryopreserved parathyroid tissue offers distinct advantages over alternative techniques.