The introduction of RT-PCR made it possible to detect circulating tumor cells in melanoma patients by analysis of melanoma-associated transcripts, especially tyrosinase. Since the development of the first PCR method for tyrosinase mRNA, several studies have presented varying results. In the present thesis I have developed and used quantitative PCR methods in order to evaluate methodological and biological factors which may explain the disparity in the literature.
Two methods were developed for tyrosinase, one competitive PCR and one real-time PCR method. With the real-time PCR technique, quantitative methods were also developed for tyrosinase related protein (TRP)-1, TRP-2, MART-1/Melan-A, S-100, GD2 synthase, MAGE-A3 and MAGE-A12. Methodological studies on the RNA extraction showed that the silica based RNA extraction method QIAamp gave considerably higher yields compared with the phenol-chloroform based extraction methods Ultraspec and FastRNA. Further studies showed that yields comparable with the QIAamp method could be obtained with the mRNA extraction method GenoPrep. Optimization of the cDNA synthesis procedure revealed that the reverse transcriptase and factors in the RNA sample inhibited the following PCR. This was avoided by diluting the cDNA sample before PCR.
The stability of the tumor cell RNA in the samples is of great concern when it comes to transporting samples from distant hospitals to the laboratory. It was found that blood collected in ACD was best, although insufficiently, stabilized when stored at +4 °C compared with room temperature. Similar stability was also obtained for PAXgene tubes stored at room temperature, however the stability of RNA was much improved when the PAXgene tubes were frozen.
Studies on the biological variation in cultured melanoma cell lines and tissue sections from melanoma metastases showed that the expression of melanoma associated transcripts varied widely. In melanoma cell lines the expression of the transcripts tyrosinase, TRP-1, TRP-2 and MART-1/Melan-A was related to the pigmentation of the cell lines. The pigment-related transcripts and S-100 were expressed at higher levels than GD2 synthase, MAGE-A3 and MAGE-A12 in the melanoma metastases. The expressions of TRP-2 and GD2 synthase appeared to be influenced by therapy. In metastases from patients treated with a combination of cisplatinum, DTIC and interferon-α2b, TRP-2 was expressed at higher levels and GD2 synthase at lower levels compared with untreated patients.
Linköping: Linköpings universitet , 2004. , 61 p.
2004-04-07, Aulan, Administrationshuset, Hälsouniversitetet, Linköping, 09:00 (Swedish)