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- Malec, Maria
(författare)
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Standardization and application of quantitative PCR methods in patients with hematological malignancies
- 2004
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Using modem combination chemotherapy regimens, it has been possible to increase the rate of complete remission (CR) in acute lymphoblastic (ALL) and acute myeloid leukemia (AML). The major reason for treatment failure in acute leukemia (AL) is the resistance of leukemic blasts to chemotherapeutic agents, which is accompanied by an inability of the immune system to eradicate residual leukemic cells surviving initial treatment. Such cells are referred to as minimal residual disease (MRD), and are very difficult to detect against the background of regenerating bone marrow (BM). The clinical definition of CR is traditionally based on conventional cytomorphological techniques, which have detection limits of 1-5%. Therefore more sensitive methods are needed for accurate evaluation of the presence of MRD. This thesis has been aimed at the standardization and assessment of molecular techniques for MRD detection in AL. A further goal has been the development of similar methods to gain quantitative information on the mRNA expression of various cytokines in lymph node biopsies from patients with Hodgkin's lymphoma (HL). Experience from the cooperative projects "BIOMED-1 Concerted Action" (CA) and "Europe Against Cancer" (EAC) contributed to MRD evaluation in a standardized way. The collaborative BIOMED-1 study resulted in the standardization of primer sets, and conditions for RT-PCR, firstly for its use in clinical practice for the molecular classification of AL at diagnosis, and secondly for MRD detection during follow-up, in order to evaluate treatment effectiveness. The collaborative efforts of EAC resulted in the development of primers and probes for real-time quantitative (RQ)-PCR analysis for fusion gene (FG) and control gene (CG) transcripts (ABL, GUS, B2M). We applied RQ-PCR to investigate the differences over time in the stabilities of FG and CG transcripts, which may result in over- or under-estimation of MRD levels. The mean stability of most FG transcripts seemed to be comparable to those of the CG; however large differences were observed between patients expressing the same FG transcripts. To compare MRD levels in follow-up BM samples from children with ALL two main methods were used: FC and semi-quantitative ASO-PCR techniques. Comparison between FC and ASOPCR showed significantly consistent results in 78% of the samples. Development of RQ-PCR permitted accurate quantification of PCR products during the exponential phase of the PCR amplification process, in complete contrast to classical PCR end-point quantification. We applied RQ-PCR and FC methods, to compare MRD levels in follow-up samples from 22 ALL patients. When results obtained by RQ-PCR and FC were grouped into positive-negative categories, a significant level of agreement was found in 72% of samples. However, if a cutoff level of 0.01% was applied, the concordance was 89%. Application of RQ-PCR techniques gains quantitative information on DNA and mRNA levels, and so we applied this approach to investigate cytokine mRNA expression in Hodgkin's Lymphoma (HL) samples and HL cell line. Reactive lymphatic tissue and peripheral blood mononuclear cells (PBMCs) from healthy donors were used as controls. The results demonstrated that expression levels of IL-13, IL-10, IL-1beta, IFNgamma, IL-15, and IL-12p35 in HL tissue were higher than those in PBMCs and reactive tissue. The highest mRNA levels for several of the cytokines were found in Epstein-Barr virus-positive samples. In conclusion, this study provides the basis for rational and efficient molecular quantitative determination of MRD levels. These approaches are conducted whilst the patient is within the first months of treatment, and give information, which allows patient classification into low or high-risk groups. These groups may benefit from the possibility for group-specific treatment modalities, which minimize side effects and optimize treatment outcome. Moreover, the application of these methods can give valuable information on inherent biological variation between different lymphoma samples, and may help to unravel the complex cytokine network contributing to the clinical and biological heterogeneity of these diseases.
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- Thörn, Ingrid, 1957-, et al.
(författare)
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Analysis of IG/TCR gene rearrangements in Swedish childhood acute lymphoblastic leukemia diagnosed 2002-2006: a multi-centre study supporting the applicability of real-time-PCR for minimal residual disease assessment
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Annan publikation (övrigt vetenskapligt/konstnärligt)abstract
- Minimal residual disease (MRD) detection during the early treatment phase has become an important stratification parameter in many childhood acute lymphoblastic leukemia (ALL) treatment protocols. Here we aimed to address the applicability of real-time quantitative polymerase chain reaction (RQ-PCR) of rearranged antigen receptor genes as an MRD method in a multi-centre setting. From a Swedish population-based cohort of 334 ALL cases diagnosed during 2002-2006, we analysed 279 diagnostic samples (84%) by screening for rearranged immunoglobulin (IG) and T-cell receptor (TCR) genes. Allele-specific oligonucleotides were designed for each patient rearrangement, and the sensitivity and quantitative level was determined for each target. The analyses were performed at five different centres while interpretation of the results was performed at consensus meetings. Overall, clonal IG/TCR rearrangements were detected in 97% (236/244) of B-cell precursor ALL (BCP ALL) and 94% (33/35) of T-ALL. A sensitive RQ-PCR analysis (≤ 10-4) was obtained in 89% (216/244) of BCP ALL and in 74% (26/35) of T-ALL. With the stratification threshold of ≥10-3 for identification of high-risk patients, 93% of BCP ALL and 86% of T-ALL reached this quantitative range by at least one target gene. Taken together, this national multi-centre study supports the use of RQ-PCR analysis as a robust method for MRD detection in the majority of childhood ALL cases.
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- Thörn, Ingrid, et al.
(författare)
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Applicability of IG/TCR gene rearrangements as targets for minimal residual disease assessment in a population-based cohort of Swedish childhood acute lymphoblastic leukaemia diagnosed 2002-2006.
- 2010
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Ingår i: European journal of haematology. - : Wiley. - 1600-0609 .- 0902-4441. ; 84:2, s. 117-27
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Tidskriftsartikel (refereegranskat)abstract
- Minimal residual disease (MRD) detection during the early treatment phase has become an important stratification parameter in many childhood acute lymphoblastic leukaemia (ALL) treatment protocols. Here, we aimed to address the applicability of rearranged antigen-receptor genes as potential MRD markers using real-time quantitative polymerase chain reaction (RQ-PCR) in a Swedish population-based cohort. From 334 childhood ALL cases diagnosed during 2002-2006, we analysed 279 diagnostic samples (84%) by screening for rearranged immunoglobulin (IG) and T-cell receptor (TCR) genes. Allele-specific oligonucleotides were designed, and the sensitivity and quantitative level was determined for each target. Overall, clonal IG/TCR rearrangements were detected in 97% (236/244) of B-cell precursor ALL (BCP ALL) and 94% (33/35) of T-ALL. A sensitive RQ-PCR analysis (< or = 10(-4)) was obtained in 89% (216/244) of BCP ALL and in 74% (26/35) of T-ALL, whereas two sensitive targets were only available in 47% (115/244) of BCP ALL and 29% (10/35) of T-ALL cases. With the stratification threshold of > or = 10(-3), which is applied in the current Nordic treatment protocol (NOPHO-ALL 2008) for the identification of high-risk patients, 93% of BCP ALL and 86% of T-ALL reached this quantitative range by at least one target gene. Taken together, this national retrospective study demonstrates that an IG/TCR target for MRD monitoring can be identified in the majority of childhood ALL cases, whereas identification of a second sensitive target gene needs to be improved.
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- Thörnerup, Ingrid, et al.
(författare)
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Minimal residual disease assessment in childhood acute lymphoblastic leukaemia: a Swedish multi-centre study comparing real-time polymerase chain reaction and multicolour flow cytometry.
- 2011
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Ingår i: British Journal of Haematology. - : Wiley. - 0007-1048 .- 1365-2141. ; 152:6, s. 743-753
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Tidskriftsartikel (refereegranskat)abstract
- Minimal residual disease (MRD) assessment is a powerful prognostic factor for determining the risk of relapse in childhood acute lymphoblastic leukaemia (ALL). In this Swedish multi-centre study of childhood ALL diagnosed between 2002 and 2006, the MRD levels were analysed in 726 follow-up samples in 228 children using real-time quantitative polymerase chain reaction (RQ-PCR) of rearranged immunoglobulin/T-cell receptor genes and multicolour flow cytometry (FCM). Using an MRD threshold of 0·1%, which was the sensitivity level reached in all analyses, the concordance between RQ-PCR and FCM MRD values at day 29 was 84%. In B-cell precursor ALL, an MRD level of ≥0·1% at day 29 predicted a higher risk of bone marrow relapse (BMR) with both methods, although FCM was a better discriminator. However, considering the higher median MRD values achieved with RQ-PCR, a higher MRD cut-off (≥0·2%) improved the predictive capacity of RQ-PCR. In T-ALL, RQ-PCR was notably superior to FCM in predicting risk of BMR. That notwithstanding, MRD levels of ≥0·1%, detected by either method at day 29, could not predict isolated extramedullary relapse. In conclusion, the concordance between RQ-PCR and FCM was high and hence both methods are valuable clinical tools for identifying childhood ALL cases with increased risk of BMR.
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