ABSTRACT
Extraocular dissemination is the main cause of death in patients with retinoblastoma (RB) in developing countries, and there are few molecular markers that are useful for the evaluation of minimal disseminated disease. The GD2 ganglioside is known to be expressed by RB cells that metastasize in bone marrow, and the activity of the enzyme responsible for its synthesis, GD2 synthase, can be detected in neuroblastoma, which shares many phenotypic features with RB. The purpose of the present study was to optimize the detection of GD2 synthase expression by reverse transcription-polymerase chain reaction (RT-PCR) followed by nested-PCR in human RB cell lines and patient samples. The optimization strategy was carried out using the RB cell lines Y79 and WERI-Rb1 and specific primers designed for the human sequence of GD2 synthase mRNA. We detected GD2 synthase expression with at least 200 and 40 pg of total RNA extracted from cultured RB cells using a first round of RT-PCR amplification or a second round of nested-PCR, respectively. We also confirmed the expression of GD2 synthase by RT-PCR and immunohistochemical detection of the ganglioside in human RB tumors xenotransplanted in nude mice. Using tumor bank specimens from eight RB patients, we were able to demonstrate the presence of GD2 synthase mRNA in blood and cerebrospinal fluid samples in cases of extraocular dissemination of the tumor. The sequence was not detected in samples derived from children with low-risk disease or healthy adult volunteers. Hence, GD2 synthase mRNA detection through an optimized nested RT-PCR assay is a promising tool for the assessment of minimal disseminated disease in enucleated patients.
ABSTRACT
INTRODUCTION: Silver stain of reticular fibres demonstrates the fine structure of the cardiac collagen network. However, nuclei are also stained with current techniques, a drawback that makes computer image analysis difficult. To solve this problem our study was designed to modify Gomori's method. Reactive concentrations and action times represent the core of that modification. Only stromal tissue is stained. The technique was tested for repeatability and reproducibility to assess the precision of the measurement procedure in the assessment of myocardial collagen in a consecutive series of myocardial samples from patients with and without heart muscle disease. In addition, we checked the reliability of the method by comparing our results with the point-counting method (PCM). METHODS: The right ventricular myocardium was fixed in 10% buffered formaldehyde and routinely processed. Paraffin sections (4 microm) were stained with several modifications of Gomori's method. Variable concentrations of potassium permanganate, silver solutions and different oxidation times were tried. A field from each sample was digitized. Additionally, the technique was tested for repeatability and reproducibility. RESULTS: We obtained absence of background and nuclear staining together with a highly contrasted image of the collagen network with 3 min oxidation with 2% potassium permanganate and a silver concentration of 1% during 5 min. In this way, it was very easy to perform acquisition, thresholding and area measurement without any further manual processing of the image. CONCLUSIONS: This technique appears very helpful for the quantitative study of the cardiac collagen network by means of computerized image analysis systems.
