Paraffin-embedded tissue as a source of RNA for gene expression analysis in oral malignancy

OBJECTIVE: To assess the feasibility of using archival oral mucosal tissue to examine gene expression at the ribonucleic acid (RNA) level.
MATERIALS AND METHODS: We describe the isolation of RNA from 8 nm sections of formalin-fixed paraffin-embedded oral mucosal tissue. RNA was reverse transcribed and three candidate genes amplified by polymerase chain reaction (PCR). The ribosomal protein S14 gene is a housekeeping gene which has been used as an internal standard in several quantitative PCR protocols. The thymidine kinase (TK) gene is expressed at low levels in most tissues and, with a well-documented genomic organisation, is a useful tool for discrimination between genomic DNA and cDNA. The RIa gene is reported to be overexpressed in many cancer cell lines, in malignant tissue and in vitro transformed cellS. RESULTS: The S14 gene, the TK gene and the RIα gene of the cAMP-dependent protein kinase (PKA) were amplified successfully from formalin-fixed paraffin-embedded tissue sections. The TK primer pair is a useful additional tool in the unambiguous identification of RNA-derived species.
CONCLUSION: RNA suitable for reverse transcribed (RT)-PCR was extracted from archival oral mucosal tissue. This should permit rapid sequence analysis of transcribed tumor suppressor genes and oncogenes in this material. Furthermore, the RT-PCR approach described may allow quantification of gene expression in oral mucosal archival material processed in a standard fashion.     

Topical 5-aminolevulinic acid mediated photodynamic therapy of superficial basal cell carcinoma using two light fractions with a two-hour interval: long-term follow-up

Photodynamic therapy (PDT) of superficial basal cell carcinoma using topical 5-aminolaevulinic acid (ALA) and 75-100 J/cm2 light dose yields unsatisfactory long-term results. In several animal models, illumination with two light fractions approximately 2 h apart was considerably more effective than single illumination, suggesting the need for a pilot clinical study. Fifteen patients with a total of 86 primary superficial basal cell carcinomas, received topical ALA and were illuminated 4 and 6 h later, both with 45 J/cm2 laser light (633+/-1 nm). Fluorescence spectra were measured before and immediately after each illumination. At a mean follow-up of 59 months (range 44-82), 67 lesions could be evaluated, 56 of which showed a complete response (84%). Cosmesis was good/excellent in 88% of the complete response group and fair in 12%. There was no correlation between protoporphyrin fluorescence and response, but a significant correlation between the percentage of fluorescence left after photobleaching by the first illumination and the amount of protoporphyrin re-synthesized 2 h later. In conclusion, the long-term complete remission rate of fractionated ALA-mediated PDT of superficial basal cell carcinoma as reported here is significantly better than after PDT with single illumination previously reported by others, but equal to studies using single illumination with a much higher light fluence. Further improvement may be possible by reducing the fluence of the first fraction, with constant total fluence.     

Early morphological changes induced by photodynamic therapy in amelanotic greene melanoma implanted in the anterior eye chamber of rabbits

Morphological changes induced by photodynamic therapy (PDT) in amelanotic Greene melanoma implanted in the anterior eye chamber of rabbits were followed up to 24 h after PDT to study the development of tissue and cell damage leading to necrosis. Immediately after PDT, blood circulation had stopped as shown by fluorescence angiography and light and electron microscopy. It was not restored during the observation period. The first signs of tumour tissue damage, shrinkage of tumour cells and enlargement of intracellular spaces, became apparent immediately after PDT. Tissue and cell destruction increased further, and 24 h after PDT the tumours were almost completely necrotic. The most intriguing finding, by electron microscopy, was the presence of mitochondria with fused membranes in the untreated melanoma cells and the dramatic increase of this aberration directly after PDT. Melanocytes and fibroblasts in the same regions did not exhibit these aberrant mitochondria and furthermore kept a normal fine structure after PDT. Artificially induced ischaemia led to swollen mitochondria with ballooned cristae but showed no increase in membrane fusions. PDT thus directly interferes with mitochondrial structure. Direct damage to tumour cells therefore presumably contributes to tumour necrosis.     

Light dosimetry in optical phantoms and in tissues: I. Multiple flux and transport theory

This is the first of two papers on the quantitative measurement of light energy fluence rates in optical phantoms and in tissues, in vitro and in vivo. The theory discussed in the present paper will be used in a forthcoming experimental paper to quantitatively check measurements of light energy fluence rates. A simple multiple flux model, which is equivalent to the diffusion approximation, is derived from the equation of transfer in a plane as well as in a spherical geometry. The equations obtained are similar to those of the Kubelka-Munk and related heuristic models. This permits conclusions regarding the limitations of these models and the values of their constants. The heuristic models are equivalent to diffusion theory for diffuse incident light, but not for collimated incident light. We also present a simple calculation of the radiance as a function of direction in the diffusion domain. This, together with the effective attenuation coefficient, permits indirect experimental determination of both the albedo and the anisotropy factor (g) of the scattering function. Similarity relations are discussed, as they result from the so called delta-Eddington approximation, leading to the conclusion that far from boundaries and sources light propagation characteristics do not change very much when g and omega s are varied, provided omega s (1-g) is kept constant (omega s = scattering coefficient). Therefore, only two optical constants are required to approximately describe light propagation in homogeneous and isotropic media in the diffusion approximation.