Fluorescence in situ hybridization as a diagnostic modality in melanocytic neoplasma.

It has well been established by comparative genomic hybridizations that melanocytic neoplasms have chromosomal copy number aberrations not seen in benign melanocytic nevi. In a rigorous study involving over 400 melanocytic neoplasms we recently evaluated the potential of a number of chromosomal loci frequently altered in melanoma but not in nevi as potential targets for a fluorescence in situ based assay. After evaluating 14 potential chromosomal loci arranged in various 4 probe panels, 6p25, 6q23, Cep6 and 11q13 were identified as the combination of targets best able to differentiate between malignant melanoma and benign nevi. Melanocytic neoplasms were considered as positive for melanoma if any of the following criteria were met; greater than 29% of enumerated cells showed gains in 6p25; greater than 38% of cells showed gains in 11q13; greater than 55% of cells show more copies of 6p25 than Cep6; or if greater than 42% of cells have less copies of 6q23 than Cep6. In a validation set, this 4 probe assay targeting these loci was able to differentiate between melanoma and nevi with a sensitivity of 86.7% and specificity of 95.4%. In this paper we review the multiple steps involved in development of this assay as well as the subsequent performance of this assay in a number of studies looking at its utility in specific differential diagnoses in melanocytic pathology.

The effect of ammonium chloride on metabolism of primary neurons and neuroblastoma cells in vitro.

Hyperammonemia is a consistent finding in many metabolic disorders. The excess ammonia (NH4Cl) interferes with brain energy metabolism possibly in part by inhibiting the tricarboxylic acid (TCA) cycle. Inhibition of the TCA cycle may result in depletion of ATP in the brain cells. In this study, the acute and chronic effect of NH4Cl (7.5 mM and 15 mM) on the metabolism of isolated neurons and neuroblastoma cells was examined. These cells were treated with NH4Cl for 15 minutes and 24 hours. Morphologic and metabolic toxicity were greater in neuroblastoma cells than in primary neurons. Following 15 minutes treatment, concentration of lactate increased significantly in neuroblastoma cells but, the concentration of other metabolites did not change significantly in neuroblastoma cells and in primary neurons. Following 24 hours treatment, the glucose utilization increased in both cell types. This high utilization of glucose in neuroblastoma cells was in concert with an increase in lactate and decrease in glutamate and ATP. In primary neurons, following 24 hours treatment, the glucose utilization significantly increased, but the concentration of the other metabolites did not change significantly. Neuroblastoma cells consumed more glucose than primary neurons in absence of NH4Cl, but generated the same amount of lactate as neurons.

Responses in primary astrocytes and C6-glioma cells to ammonium chloride and dibutyryl cyclic-AMP.

Elevated brain ammonia levels are a major factor in the genesis of hepatic encephalopathy (HE). The mechanism of ammonium chloride (NH4Cl) neurotoxicity involves interruption of oxidative metabolism. This leads to decreased levels of ATP concentration and subsequent glial fibrillary acidic protein (GFAP) degradation of astrocytes and fibrous C6-glioma cells. Our study investigates NH4Cl toxicity by evaluating changes in ATP concentration and mitochondrial function as well as by evaluating alterations in GFAP expression. NH4Cl induced decreases in ATP were detected after 15 minutes in C6-glioma cells and 24 hours in both cell types. Mitochondrial function, assessed by MTT (2-4,5-dimethylthiazol A-yl)-2, 5-diphenyltetrazolium bromide) assay, was impaired in both cell types at 24 hours following NH4Cl treatment. GFAP was also significantly decreased in both cell types. Morphologic and metabolic toxicities were greater in C6-glioma cells. The data clearly indicate that NH4Cl interrupts oxidative metabolism. The greater toxicity seen in C6-glioma cells may be due to their greater dependence on oxidative metabolism. Lastly, the decrease in GFAP is probably a consequence of diminished ATP.