Jeffrey C. Pence

c-erbB-2 Expression in Breast Cancer Detected by Immunoblotting and Immunohistochemistry

Evidence that the c-erbB-2 proto-oncogene is important in prognosis and oncogenesis in a number of human malignancies is increasing. DNA (Southern) hybridization and immunoblotting (Western) techniques are most commonly utilized to determine the amplification and protein expression of this proto-oncogene, respectively. These extraction techniques are often time consuming, costly, and subject to variability depending on the histological characteristics of the tumor. Immunohistochemistry (IHC), on the other hand, is more often time and cost effective. In addition, IHC may offer enhanced sensitivity over extraction techniques because of the in situ nature of analysis. In data presented here, 71 cases of human mammary carcinoma were concomitantly assessed for c-erbB-2 gene copy number and oncoprotein expression by dilution DNA hybridization and IHC, respectively. In 65 (92%) of 71 cases, high-level expression was associated with gene amplification, whereas moderate or low-level expression was associated with a normal diploid gene copy number. In five of the six discrepant cases, IHC predicted amplification which was not corroborated by Southern analysis. In these cases, tumor mass was limited by the intraductal component of the lesion or by an abundance of stromal elements within the specimen. In 39 of the 71 total cases, Western immunoblotting was compared with IHC in the assessment of oncoprotein expression. Concordance was found in 33 (85%) of 39 cases. In four of the six discrepant cases, high levels of c-erbB-2 expression were demonstrated by IHC but not by immunoblotting. In these cases, intraductal disease and stroma-rich tumors again led to a relative paucity of neoplastic tissue within the specimens. We conclude that IHC offers a favorable alternative to either Southern analysis or Western immunoblotting in the assessment of c-erbB-2 gene copy number and expression levels of oncoprotein in human mammary carcinoma. Furthermore, IHC may prove advantageous to either extraction technique in specimens with limited tumor mass, such as biopsy materials, stroma-rich tumors, or early stage lesions such as intraductal carcinoma.

Effects of Pulsatile Perfusion on Human Saphenous Vein Vasoreactivity: A Preliminary Report

This study examined the effects of exposure to arterial blood pressure and flow on human saphenous vein catecholamine sensitivity. Unused portions of saphenous vein from eight patients undergoing peripheral bypass procedures were mounted parallel in a specially designed organ culture apparatus and perfused with tissue culture medium with 95% CO(2) at 37 degrees C. One segment was fixed between two cannulas while the medium was gently agitated (control) and the other was actively perfused via a pulsatile pump system at a rate of 60 beats/min, peak pressure of 100 mmHg and peak flow of 200 ml/min (pulsed; mean pressure 60 mmHg; mean flow 115 ml/min). After 48 h, vein segments were removed and tested for in vitro isometric contraction in response to KCI, norepinephrine and histamine, and relaxation in response to acetylcholine, calcium ionophore A23187, and sodium nitroprusside. There were no differences in mean(s.e.m.) maximal contraction in response to KCI (control 0.61(0.16) g versus pulsed 0.72(0.27)g; P = n.s.), norepinephrine (control 1.00(0.56) g versus pulsed 1.51(0.54) g; P= n.s.), or histamine (control 1.47(0.85) g versus pulsed 1.95(0.64) g; P= n.s.). However, pulsed veins exhibited increased sensitivity to both norepinephrine (control -logED50 6.20(0.23) versus pulsed mean(s.e.m.) 6.60(0.17); P< 0.05) and histamine (control -logED(50) 5.60(0.27) versus pulsed 6.24(0.20); P = 0.05). Pulsed veins exhibited slightly less acetylcholine-induced relaxation although the difference did not reach statistical significance (control mean(s.e.m.) relaxation at 1 x 10(6)M 9.2(14.0)% versus pulsed -13.3(6.4)%; P = n.s.). There were no differences in relaxation in response to either A23187 (control 1 x 10-(4)M 178(19)% versus pulsed 191(68)% or sodium nitroprusside (control 225(15)% versus pulsed 254(17)%; P = n.s.). The data presented herein indicate that exposure of human saphenous vein to the hemodynamics of the arterial environment for 48 h results in catecholamine supersensitivity while contractile and relaxant function are not affected.

Autoregulation of Neuroblastoma Growth by Vasoactive Intestinal Peptide

Elevated serum levels of vasoactive intestinal peptide (VIP) are associated with some cases of neuroblastoma and correlate with a favorable prognosis. VIP has previously been shown in our laboratory to cause the in vitro growth inhibition and morphological differentiation of the human neuroblastoma cell line, LA-N-5. It is now shown that LA-N-5 cells express immunoreactive VIP and bear specific VIP receptors. Antagonism of endogenous VIP, either by competitive inhibition or receptor blockade, increased cell proliferation, suggesting that VIP is operative in normal growth regulation. Intracellular and extracellular levels of VIP were also shown to increase significantly during the retinoic acid-induced differentiation of these cells. Furthermore, a concomitant marked increase in VIP receptor expression was demonstrated with cellular differentiation. These receptors remain functional as evidenced by a matching increase in the level of detectable cAMP generated in response to exogenous VIP. It is concluded that VIP is a normal autoregulator of neuroblastoma cell growth and differentiation, and that retinoic acid-mediated differentiation may be, in part, due to endogenous VIP.

Retinoic acid-induced regulation of neuropeptide Y receptor expression and function in the neuroepithelioma line SK-N-MC

UNLABELLED Neuropeptide Y (NPY) acts through specific receptors to inhibit adenyl cyclase and may have a role in neuroblastomas and neuroepitheliomas (NE) as a regulator of cell growth and differentiation. The authors have examined the status of NPY receptors in the NE cell line SK-N-MC and the effect of retinoic acid (RA), a known differentiating agent, on their expression and function. METHODS Competitive NPY binding studies were performed on normal and RA-treated cells, followed by Scatchard analysis. NPY receptor function in the absence of or following RA treatment was determined by the ability of various concentrations of NPY to attenuate the forskolin-stimulated accumulation of intracellular cAMP. The mitogenic effect of NPY was evaluated by growing normal or RA-treated cell in the presence of various concentrations of NPY. RESULTS Scatchard analysis showed a Kd of 2.3 nmol/L and a Bmax of 91,000 receptors per cell for untreated cells. RA treatment decreased receptor expression to 11,700 per cell without a significant change in receptor affinity (3.6 nmol/L). The effect of forskolin was inhibited by NPY in a dose-dependent fashion in both untreated and treated cells indicating functional receptors in both NPY stimulates the growth of normal SK-N-MC cells. NPY stimulated growth was significantly attenuated after RA treatment, possibly as a result of decreased NPY receptor expression. CONCLUSIONS Treatment of SK-N-MC cells with RA, a known differentiating agent, leads to decreased expression of functional NPY receptors and a concomitant decrease in the mitogenic effect of NPY. This supports the role for NPY in the pathogenesis of NE.

DNA Ploidy of Basal Cell Carcinoma Determined by Image Cytometry of Fresh Smears

Image analysis of nuclear DNA content (DNA ploidy) was performed on smears of basal cell carcinoma (BCC) obtained during Mohs microscopically‐controlled surgery from 51 tumors. DNA ploidy was compared with histologic growth pattern and the contour of the invading edge. There was a statistically significantly increased frequency of DNA aneuploidy in smears from BCC exhibiting partial or total diffuse (infiltrative and superficial multicentric) growth patterns (80%; 32 of 40) as compared to solely circumscribed growth patterns (0%; 0 of 11) (p< 0.001).

Detection of a Novel Marker in the Bronchial Secretions of Patients with Non-Small Cell Lung Cancer using the 4B5 Monoclonal Antibody

A murine monoclonal antibody designated 4B5 was raised against the high molecular weight fraction of pooled sputum from patients with non‐small cell lung cancer (NSCLC). Immunohistochemical staining indicated that 4B5 binds to histologically normal bronchial epithelium distant from tumor in 72% (39 of 54) of patients with NSCLC, but it binds to the primary cancer in only 13% (7 of 54) of the same patients. The antibody reacted less intensely with the bronchial epithelium in 16.6% (3 of 18) of autopsied patients without significant lung disease. The antigen recognized by 4B5 is a high molecular weight glycoprotein of more than 400 kilodaltons, judged by gel filtration and sodium dodecyl sulfate‐poly‐acrylamide gel electrophoresis and western blot analysis. Antigenic activity persisted after heating and resisted treatment with neuraminidase, but it was destroyed using protease and periodate. Multiple epitopes were present on each molecule recognized by 4B5. The determinants recognized by this antibody deserve additional study as possible markers of premalignant change in patients with NSCLC.

Diffuse Infantile Hemangiomatosis of the Ileum Presenting with Multiple Perforations: A Case Report and Review of the Literature

Hemangiomas of the small intestine are rare, accounting for only 0.05% of all intestinal neoplasms (Jarvi et al. J Pediatr Gastroenterol Nutr. 2008;46:593-597). The jejunum is the most common site of involvement in the small intestine (Levy et al. Am J Roentgenol. 2001;177:1073-1081). Small bowel hemangiomas are most commonly manifested by gastrointestinal bleeding, abdominal pain, obstruction, or intussusception. There are very few reported cases in the literature of hemangiomatosis presenting with perforation, and only 1 previously reported case of perforation in the ileum. We present a rare case of a 5-week-old female with diffuse hemangiomatosis of the ileum presenting with multiple ileal perforations and peritonitis.