Expression of Epidermal Growth Factor Receptors in Human Breast Cancer: Mass Versus Ligand Binding Capacity

Abstract: Thirty percent of lymph node-negative women with primary breast cancers are at high risk for early recurrence of metastatic disease and diminished survival. Identification of these high-risk patients is a long-term objective of our laboatory, and the epidermal growth factor receptor (EGFR) was investigated as a prognostic factor, EGFR was measured by a ligand binding assay developed in house, in the competition mode that served as the "gold standard" for assessing receptor content and activity. In contrast, measurements of mass (content) were performed by an enzyme immunoassay (EIA) from Ciba Corning Diagnostics (Alameda, CA) and by an enzyme-linked immunosorbent assay (ELISA) from Oncogene Science (Uniondale, NY). A total of 78 breast carcinomas were examined. The median binding capacity measured with [¹²⁵I]EGF was 13 fmol/mg membrane protein (range 0–981), that measured by EIA was 8 fmol/mg (range 1–125), while EGFR measured by ELISA was 135 (range 0–751). Distribution of EGFR did not appear to vary as a function of patient age. Neither the results from EIA nor those from ELISA correlated with those obtained by the ligand binding assay. However, there was a good correlation of results obtained by the two antibody-based assays despite the fact that the calibration of standards was considerably different. These data suggest that EGFR should be measured by ligand binding assay for clinical studies; mass assays based on antibody reagents will require further development.     

Postnephrectomy arteriovenous fistula of the renal pedicle treated with detachable balloons: A case report

A case of postnephrectomy arteriovenous fistula of the right renal pedicle is reported here. The diagnosis was confirmed by angiography, and successful treatment was achieved using detachable balloon.     

Natural history of atopic dermatitis and its relationship to serum total immunoglobulin E in a population-based birth cohort study

We investigated the natural history of atopic dermatitis (AD) in a population-based birth cohort and assessed whether children at risk of visible eczema at 5 years of age can be identified from total immunoglobulin E (IgE) levels measured at 8, 12 and 18 months. AD data collected included a whole body examination for visible eczema at 49 months (4 years) and 61 months (5 years) of age and parent completed questionnaire data throughout their early lives. Children were divided into four groups based on their natural history of early AD: persistent (AD at 1, 6, 18, 30 and 42 months, n  = 34), intermittent early onset (before 18  months of age, n  = 495), intermittent late onset (18–42 months of age, n  = 273) and unaffected ( n  = 429). Visible eczema at 5 years of age was present in 12.2% (117/957) (95% confidence interval [CI] 10.1–14.3%) of the children. Levels of total IgE at 8, 12 and 18 months of age were associated with early onset of AD, but not with AD of later onset. For all four natural history groups, the geometric mean total IgE at 12 months was higher in those who subsequently had visible eczema than those who did not. However, the degree of overlap was such that total IgE at 12 months of age was a poor predictor of eczema at age five. A cutoff point of 78 kU/l had the highest positive predictive value for visible eczema at 5 years of age of 28.6%, with a sensitivity of 12% and specificity of 95%.     

Periosteal remodeling at the femoral neck in nonhuman primates.

Periosteal bone turnover is poorly understood. We documented intramembranous periosteal bone turnover in the femoral neck in intact nonhuman primates and an increase in osteoclast numbers at the periosteal surface in sex steroid-deficient animals. Our studies are the first to systematically document periosteal turnover at the femoral neck. INTRODUCTION: Bone size is an important determinant of bone strength, and cellular events at the periosteal surface could alter bone dimensions. We characterized periosteal cellular activity with dynamic histomorphometric studies of nonhuman primate femoral neck and shaft. MATERIALS AND METHODS: Femur specimens from 16 intact adult male and female nonhuman primates (Rhesus [Macaca mulatta, n = 9] and Japanese Macaque [Macaca fuscata, n = 7]) were analyzed. Animals were double-labeled with tetracycline, and necropsy was performed 2-7 days after the last dose. We characterized periosteal resorptive activity in an additional group of five intact and four castrate female animals. Multiple group comparisons in intact animals were performed by one-way ANOVA followed by a Fisher PLSD posthoc test. In gonadectomized animals, Fisher's exact test was used for dichotomous and Mann-Whitney U-test for continuous variables. RESULTS: Bone turnover in the periosteum of the femoral neck in intact animals was more rapid than at the femoral shaft but slower than in femoral neck cancellous bone. Similarly, in these intact animals, the eroded surface of cortical bone at the femoral neck periosteal surface was significantly greater than in the cancellous bone compartment (p < 0.0001) or on the femoral shaft (p < 0.0001). Gonadectomized female animals showed an increase in osteoclast number on the periosteal surface compared with intact controls (p < 0.01). CONCLUSIONS: We documented intramembranous periosteal bone turnover in the femoral neck by histomorphometric analyses. The tissue level bone formation rate was sufficient to add substantively to femoral neck size over time. Periosteal osteoclastic activity was not the result of the emergence of intracortical tunneling at the bone surface. Sex steroid deficiency produced an increase in osteoclast numbers at the periosteal surface. This is the first systematic documentation of periosteal turnover at the femoral neck.     

Internalization of sst2, sst3, and sst5 receptors: effects of somatostatin agonists and antagonists.

The uptake of radiolabeled somatostatin analogs by tumor cells through receptor-mediated internalization is a critical process for the in vivo targeting of tumoral somatostatin receptors. In the present study, the somatostatin receptor internalization induced by a variety of somatostatin analogs was measured with new immunocytochemical methods that allow characterization of trafficking of the somatostatin receptor subtype 2 (sst2), somatostatin receptor subtype 3 (sst3), and somatostatin receptor subtype 5 (sst5) in vitro at the protein level. METHODS: Human embryonic kidney 293 (HEK293) cells expressing the sst2, sst3, or the sst5 were used in a morphologic immunocytochemical internalization assay using specific sst2, sst3 and sst5 antibodies to qualitatively and quantitatively determine the capability of somatostatin agonists or antagonists to induce somatostatin receptor internalization. In addition, the internalization properties of a selection of these agonists have been compared and quantified in sst2-expressing CHO-K1 cells using an ELISA. RESULTS: Agonists with a high sst2-binding affinity were able to induce sst2 internalization in the HEK293 and CHO-K1 cell lines. New sst2 agonists, such as Y-DOTA-TATE, Y-DOTA-NOC, Lu-DOTA-BOC-ATE (where DOTA is 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid; TATE is [Tyr3, Thr8]-octreotide; NOC is [1-NaI3]-octreotide; and BOC-ATE is [BzThi3, Thr8]-octreotide), iodinated sugar-containing octreotide analogs, or BIM-23244 were considerably more potent in internalizing sst2 than was DTPA-octreotide (where DTPA is diethylenetriaminepentaacetic acid). Similarly, compounds with high sst3 affinity such as KE108 were able to induce sst3 internalization. In sst2- or sst3-expressing cell lines, agonist-induced receptor internalization was efficiently abolished by sst2- or sst3-selective antagonists, respectively. Antagonists alone had no effect on sst2 or sst3 internalization. We also showed that somatostatin-28 and somatostatin-14 can induce sst5 internalization. Unexpectedly, however, potent sst5 agonists such as KE108, BIM-23244, and L-817,818 were not able to induce sst5 internalization under the same conditions. CONCLUSION: Using sensitive and reproducible immunocytochemical methods, the ability of various somatostatin analogs to induce sst2, sst3, and sst5 internalization has been qualitatively and quantitatively determined. Whereas all agonists triggered sst2 and sst3 internalization, sst5 internalization was induced by natural somatostatin peptides but not by synthetic high-affinity sst5 agonists. Such assays will be of considerable help for the future characterization of ligands foreseen for nuclear medicine applications.     

Permeability of 13 different gloves to 13 cytotoxic agents under controlled dynamic conditions.

PURPOSE: The permeability of 13 different gloves to 13 cytotoxic agents under controlled dynamic conditions is described. METHODS: Thirteen cytotoxic agents were prepared at the highest concentrations normally encountered by pharmacy personnel. Four glove types--neoprene, natural rubber latex, nitrile, and vinyl--were exposed to the cytotoxic agents for 15, 30, and 60 minutes. Tests were conducted using the middle finger of each glove. Linearity, reproducibility, and sensitivity were evaluated for each drug tested. Assays were run using liquid chromatographic tandem mass spectrometry (LC/MS/MS) and high-performance liquid chromatography with ultraviolet light (HPLC-UV). Permeability testing was conducted using an original system designed to evaluate dynamic constraints, such as rubbing, stretching, and tension. RESULTS: Linearity by LC/MS/MS and HPLC-UV was confirmed at concentrations up to 1000 ng/mL for all drugs. Most glove materials were permeable at rates below ASTM recommendations over the one-hour testing period. Vinyl was the most permeable material. Carmustine permeated the widest variety of materials. Due to the high sensitivity of the analytic methods, all materials displayed low but significant permeability for at least one drug after one hour. Higher resistance to permeation was recorded for all neoprene, some natural rubber latex, and one nitrile glove. CONCLUSION: Neoprene, natural rubber latex, and nitrile gloves displayed the highest resistance to permeation of the 13 cytotoxic agents studied. Additional factors, such as duration of exposure, glove thickness, and drug liposolubility and molecular weight, also affected permeability.     

Preliminary experience with inhaled milrinone in cardiac surgery

Background: Inhaled administration of milrinone reduces pulmonary artery pressure. Pulmonary hypertension (PH) and right heart failure are associated with difficult separation from cardiopulmonary bypass (CPB). Therefore, inhaled milrinone could facilitate separation from CPB. Objective: To determine the impact and timing of administration of inhaled milrinone. Methods: A retrospective analysis of our experience on high-risk patients receiving inhaled milrinone was conducted to evaluate the postoperative course after administration of the drug. Results: Seventy-three patients received inhaled milrinone from June 2002 to February 2005. Mean age was 64 ± 13 years, with a mean preoperative Parsonnet score of 27 ± 14. Inhaled milrinone (5 mg) was administered before (n = 30) or after (n = 40) CPB, three patients had off-pump procedures and were excluded. CPB time was 145 ± 78 min with cross-clamping times of 91 ± 56 min without any significant difference between groups. Fifty-four patients (74%) had difficult separation from CPB, 14 patients (19%) required an intra-aortic balloon pump and 10 patients (14%) needed emergency reinitiation of CPB for hemodynamic instability. Ten patients died in the perioperative period (13.7%). Patients receiving inhaled milrinone prior to CPB initiation had a lowering pulmonary artery pressure after CPB (p < .01) and had less emergency reinitiation of CPB after weaning (3% vs 23%, p = .02) as compared to those with administration after CPB. No detectable side effects were directly linked to the administration of the drug. Conclusion: In this high-risk cohort, use of inhaled milrinone was well tolerated. Administration before initiation of CPB could help weaning from CPB.