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Expression of tumor associated transcripts in malignant melanoma metastases: with methodological aspects
Linköping University, Department of Biomedicine and Surgery, Clinical Chemistry. Linköping University, Faculty of Health Sciences.
Linköping University, Department of Biomedicine and Surgery, Oncology. Linköping University, Faculty of Health Sciences.
Linköping University, Department of Biomedicine and Surgery, Oncology. Linköping University, Faculty of Health Sciences.
Östergötlands Läns Landsting, Centre for Laboratory Medicine, Department of Clinical Pathology and Cytology. Linköping University, Faculty of Health Sciences.
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(English)Manuscript (preprint) (Other academic)
Abstract [en]

Several antigens expressed in malignant melanoma are involved in the immunological surveillance of the tumor. The mRNAs of these antigens, especially tyrosinase, have also been used in the detection of minimal residual disease in the blood of melanoma patients. We have therefore analyzed the expression of tyrosinase, TRP-1, TRP-2, MART-1/Melan-A, MAGE-A3, MAGE-A12, S-100 and GD2 synthase by real-time PCR in snap frozen sections from 28 regional and systemic metastases. Treatment with cisplatinum, dacarbazine and interferon-α2b was given to 15 patients before surgery. The transcript concentrations varied widely between individual metastases. However, in general the pigment-related transcripts and that of S-100 were found at higher levels than those of MAGE-A3, MAGE-A12 and GD2 synthase. Significant correlations (p<0.001) were found between tyrosinase and MART-1/Melan-A and between MAGE-A3 and MAGE-A12. TRP-2 and GD2 synthase were both influenced by the treatment, TRP-2 expression was increased in the metastases from treated patients, whereas GD2 synthase expression was decreased. Furthermore, a decrease in tyrosinase expression was found in metastases without tumor-infiltrating lymphocytes. ln this work normalization by the section area contra normalization by housekeeping genes was also evaluated and similar results were obtained with both methods.

National Category
Medical and Health Sciences
Identifiers
URN: urn:nbn:se:liu:diva-84994OAI: oai:DiVA.org:liu-84994DiVA: diva2:563480
Available from: 2012-10-30 Created: 2012-10-30 Last updated: 2012-10-30
In thesis
1. Quantitative analysis of melanoma transcripts: with emphasis on methodological and biological variation
Open this publication in new window or tab >>Quantitative analysis of melanoma transcripts: with emphasis on methodological and biological variation
2004 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

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.

Place, publisher, year, edition, pages
Linköping: Linköpings universitet, 2004. 61 p.
Series
Linköping University Medical Dissertations, ISSN 0345-0082 ; 843
National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:liu:diva-24062 (URN)3621 (Local ID)91-7373-816-6 (ISBN)3621 (Archive number)3621 (OAI)
Public defence
2004-04-07, Aulan, Administrationshuset, Hälsouniversitetet, Linköping, 09:00 (Swedish)
Opponent
Available from: 2009-10-07 Created: 2009-10-07 Last updated: 2012-10-30Bibliographically approved

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Farnebäck, MalinHåkansson, AnnikaGustafsson, BertilKågedal, Bertil

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