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Quantitative analysis of melanoma transcripts: with emphasis on methodological and biological variation
Linköping University, Department of Biomedicine and Surgery, Clinical Chemistry. Linköping University, Faculty of Health Sciences.
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: urn:nbn:se:liu:diva-24062Local ID: 3621ISBN: 91-7373-816-6 (print)OAI: oai:DiVA.org:liu-24062DiVA: diva2:244378
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
List of papers
1. Quantitative analysis of tyrosinase transcripts in blood
Open this publication in new window or tab >>Quantitative analysis of tyrosinase transcripts in blood
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2000 (English)In: Clinical Chemistry, ISSN 0009-9147, E-ISSN 1530-8561, Vol. 46, no 7, 921-927 p.Article in journal (Refereed) Published
Abstract [en]

Background: Tyrosinase is an enzyme unique to pigment-forming cells. Methods using this transcript for detection of melanoma cells in blood have given divergent results. Quantitative analytical procedures are therefore needed to study the analytical performance of the methods.

Methods: Mononucleated cells were isolated by Percoll centrifugation. RNA was isolated by each of three methods: UltraspecTM-II RNA isolation system, FastRNATM GREEN Kit, and QIAamp RNA Blood Mini Kit. cDNA was synthesized using random hexamer primers. A tyrosinase-specific product of 207 bp was amplified by PCR. As an internal standard (and competitor) we used a 207-bp cDNA with a base sequence identical to the tyrosinase target except for a 20-bp probe-binding region. The PCR products were identified by 2,4-dinitrophenol (DNP)-labeled probes specific for tyrosinase (5′DNP-GGGGAGCCTTGGGGTTCTGG-3′) and internal standard (5′DNP-CGGAGCCCCGAAACCACATC-3′) and quantified by ELISA.

Results: The calibration curves were linear and had a broad dynamic measuring range. A detection limit (2 SD above zero) of 48 transcripts/mL of blood was obtained from a low control. The analytical imprecision was 50% and 48% at concentrations of 1775 and 17 929 transcripts/mL (n = 12 and 14, respectively). With the cell line SK-Mel 28 added to blood and RNA extracted with the Ultraspec, Fast RNA, and QIAamp RNA methods, we found (mean ± SD) 1716 ± 1341, 2670 ± 3174, and 24 320 ± 5332 transcripts/mL of blood. Corresponding values were 527 ± 497, 2497 ± 1033, 14 930 ± 1927 transcripts/mL of blood when the cell line JKM86-4 was added. One high-risk patient was followed by repeated analysis of tyrosinase transcripts in blood. The melanoma marker 5-S-cysteinyldopa in serum and urine was within reference values, but tyrosinase mRNA was slightly increased (120–168 transcripts/mL of blood). The tyrosinase mRNA increased to 1860 transcripts/mL concomitant with the increase in 5-S-cysteinyldopa; later a spleen metastasis was found.

Conclusions: The results obtained with different RNA extraction methods illustrate the importance of quantitative methods for validation of methods. The use of QIAamp RNA improved the extraction efficiency considerably. Data from a case study suggest the assay is suitable in the follow-up of patients with high risk of developing metastases.

National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:liu:diva-25119 (URN)9552 (Local ID)9552 (Archive number)9552 (OAI)
Available from: 2009-10-07 Created: 2009-10-07 Last updated: 2012-10-30
2. Quantitative analysis of tyrosinase and tyrosinase-related protein-2 mRNA from melanoma cells in blood by real-time polymerase chain reaction
Open this publication in new window or tab >>Quantitative analysis of tyrosinase and tyrosinase-related protein-2 mRNA from melanoma cells in blood by real-time polymerase chain reaction
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2000 (English)In: Melanoma research, ISSN 0960-8931, E-ISSN 1473-5636, Vol. 10, no 3, 213-222 p.Article in journal (Refereed) Published
Abstract [en]

Several studies have evaluated the use of polymerase chain reaction (PCR) amplification of tyrosinase mRNA to detect melanoma cells in blood. However, contradictory results have been obtained from different groups. We therefore have developed and validated a quantitative PCR method for tyrosinase and tyrosinase-related protein-2 (TRP-2) mRNA. An important methodological finding was that high concentrations of reverse transcriptase or RNA sample inhibited the following PCR. This could be abolished by dilution of the cDNA sample before the PCR. Standard curves with a linear range over at least five logs were obtained with dilutions of melanoma cell cDNA. Controls (RNA and cDNA) consisting of melanoma cells (1000/ml) added to blood were analysed repeatedly over 3 months, resulting in means between 880 and 1074 AU/ml. The RNA controls were stable, whereas the cDNA controls, as well as the calibrators, showed a tendency to change over time. The variation in the RNA controls was 25% for tyrosinase and 22% for TRP-2. Seven stage III-IV melanoma patients were tested for tyrosinase and TRP-2 transcripts in blood drawn from a peripheral vein and from a Port-a-cath. Tyrosinase mRNA was found in three patients (0.8-12.4 AU/ml). For TRP-2, the same amount was found in the patients as in healthy donors. No differences were seen between blood from a peripheral vein and from the Port-a-cath. We here present fast and sensitive methods for the quantification of tyrosinase and TRP-2 mRNA in blood.

National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:liu:diva-25089 (URN)10890374 (PubMedID)9520 (Local ID)9520 (Archive number)9520 (OAI)
Available from: 2009-10-07 Created: 2009-10-07 Last updated: 2012-10-30
3. Stability of blood samples for analysis of tumour cell transcripts
Open this publication in new window or tab >>Stability of blood samples for analysis of tumour cell transcripts
(English)Manuscript (preprint) (Other academic)
Abstract [en]

When mRNA expression from tumor cells is analyzed in blood, one of the greatest problems is to keep the samples stable during transfer to the laboratory. Improved systems for stabilization of transcripts therefore have to be developed.

Blood was collected in PAXgene™ Blood RNA Tubes and spiked with melanoma and neuroblastoma cells. The stability of tumor transcripts at room temperature was tested after 1, 2, 3, 5, 7, and 9 days. PAXgene tubes were also frozen at -20 °C and -70 °C for 14 days. Total RNA was extracted by PAXgene™ Blood RNA Kit and mRNA was extracted by the GenoPrep Direct mRNA Kit. Tyrosinase, MAGE, tyrosine hydroxylase, and GD2 synthase mRNA was quantified by real-time PCR. The stability of these transcripts was compared with the stability in ACD tubes when RNA extraction was performed with QIAamp RNA Blood Mini Kit.

The yields of transcripts from the PAXgene tubes using the PAXgene™ Blood RNA Kit varied from 37% to 49% of the yields from ACD tubes. When mRNA was extracted with the GenoPrep Direct mRNA Kit the yields increased to 67-121%. Stability tests showed significant decreases of all the transcripts in the PAXgene tubes after two days at room temperature. After seven days, 10% of the initial transcript concentrations remained, whereas 32% remained in the ACD tubes. However, in the frozen PAXgene tubes the transcript levels were unchanged for 14 days.

We conclude that the PAXgene tube is suitable when the sample is stored at -20 °C or -70 °C and mRNA is extracted using GenoPrep Direct mRNA Kit.

Keyword
RNA stability, real-time PCR, blood sampling, preanalytics, minimal residual disease
National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:liu:diva-84993 (URN)
Available from: 2012-10-30 Created: 2012-10-30 Last updated: 2012-10-30
4. Quantitative relationships between pigment-related mRNA and biochemical melanoma markers in melanoma cell lines
Open this publication in new window or tab >>Quantitative relationships between pigment-related mRNA and biochemical melanoma markers in melanoma cell lines
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2002 (English)In: Melanoma research, ISSN 0960-8931, E-ISSN 1473-5636, Vol. 12, no 3, 193-200 p.Article in journal (Refereed) Published
Abstract [en]

The use of reverse transcription polymerase chain reaction (RT-PCR) analysis of melanoma-specific transcripts for the identification of circulating melanoma cells has shown very variable results in different studies on melanoma patients. We have therefore developed quantitative methods to study both analytical and biological variations as possible causes of this phenomenon. Pigment-related and S-100β transcripts were quantified in 12 different melanoma cell lines and related to the amounts of 5-S-cysteinyldopa, pigment and S-100B protein. A real-time PCR method was used and the results were expressed as absolute number of transcripts per cell. Tyrosinase, tyrosinase-related protein (TRP)-1, TRP-2 and MART-1/Melan-A mRNA varied from undetectable (< 10-4 transcripts/cell) to 103 transcripts/cell, i.e. by a factor > 107 in the different cell lines. S-100β mRNA varied from 2.8 to 165 transcripts/cell, i.e. by a factor of 60. Tyrosinase, TRP-1 and TRP-2 mRNA correlated significantly with the amount of 5-S-cysteinyldopa, an intermediate pigment metabolite (P < 0.001, P < 0.001 and P < 0.01, respectively). The amount of S-100β mRNA correlated significantly with the amount of S-100B protein (P < 0.001). No cross-correlations were seen between the pigment-related and S-100-related analytes. We conclude that one reason behind the negative results of RT-PCR measurement of pigment-related mRNA may be that these transcripts are not always expressed in the particular cells present in the patient's blood. Furthermore, variation in the expression of the order of 107 must have great impact on the diagnostic sensitivity. Measurement of S-100β mRNA would be more sensitive, but the use of this transcript is hampered by its presence in the blood cells. © 2002 Lippincott Williams & Wilkins.

National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:liu:diva-25118 (URN)10.1097/00008390-200206000-00002 (DOI)12140375 (PubMedID)9551 (Local ID)9551 (Archive number)9551 (OAI)
Available from: 2009-10-07 Created: 2009-10-07 Last updated: 2012-10-30
5. How useful are housekeeping genes?: variable expression in melanoma metastases
Open this publication in new window or tab >>How useful are housekeeping genes?: variable expression in melanoma metastases
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2007 (English)In: Clinical Chemistry and Laboratory Medicine, ISSN 1434-6621, Vol. 45, no 11, 1481-1487 p.Article in journal (Refereed) Published
Abstract [en]

Background: There is a certain difference in opinion regarding the optimal choice of housekeeping genes used as normalization factors in gene expression analysis. We have therefore examined the suitability of three housekeeping genes, hypoxanthine phosphoribosyl transferase, β-glucuronidase and β2-micro-globulin, for normalization of expression data from melanoma metastases.

Methods: The expression of the three housekeeping genes was quantified by quantitative reverse transcription PCR in snap-frozen sections from 44 melanoma metastases, of which 19 were from patients treated with cisplatinum, dacarbazine and interferon-α2b.

Results: The expression of each housekeeping gene varied considerably between the different metastases. Histopathological examination of the tissue sections revealed variation in the amount of tumor cells in the tissue, necrosis, varying degrees of lymphocyte infiltration, and lymph node remnants. Based on this examination, 16 biopsies were omitted from further analysis because they had cracked, contained empty or necrotic areas, or were dominated by lymph node tissue. Even in sections with more than 90% tumor cells, a wide variation in the expression of the three housekeeping genes was found. The amount of lymphatic infiltrate in the tumors can have an effect on the expression of housekeeping genes in the meta-stases, whereas treatment did not seem to influence the expression.

Conclusions: We conclude that the choice of housekeeping genes can have great impact on the normalization of specific genes in melanoma metastases. Furthermore, in the analysis of mRNA expression in tumor tissue, microscopic examination is of great importance to evaluate the integrity and cellular composition of the biopsy.

National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:liu:diva-39472 (URN)10.1515/CCLM.2007.303 (DOI)48767 (Local ID)48767 (Archive number)48767 (OAI)
Available from: 2009-10-10 Created: 2009-10-10 Last updated: 2012-10-30
6. Expression of tumor associated transcripts in malignant melanoma metastases: with methodological aspects
Open this publication in new window or tab >>Expression of tumor associated transcripts in malignant melanoma metastases: with methodological aspects
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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:nbn:se:liu:diva-84994 (URN)
Available from: 2012-10-30 Created: 2012-10-30 Last updated: 2012-10-30

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