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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
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology. Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology, Hematology and Immunology. (Molecular Hematology)
Department of Clinical Sciences, Paediatrics, Umeå University, Umeå, Sweden.
Division of Hematology and Transfusion Medicine, Department of Laboratory Medicine, Lund University, Lund, Sweden.
Department of Clinical Chemistry and Transfusion Medicine, Sahlgrenska University Hospital, Gothenburg, Sweden.
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(English)Manuscript (Other academic)
Abstract [en]

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.

Keyword [en]
RQ-PCR, rearranged IG/TCR genes, minimal residual disease, childhood acute lymphoblastic leukemia
National Category
URN: urn:nbn:se:uu:diva-100936OAI: oai:DiVA.org:uu-100936DiVA: diva2:211569
Available from: 2009-04-16 Created: 2009-04-14 Last updated: 2010-01-14
In thesis
1. Minimal Residual Disease Assessment in Childhood Acute Lymphoblastic Leukemia
Open this publication in new window or tab >>Minimal Residual Disease Assessment in Childhood Acute Lymphoblastic Leukemia
2009 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Traditionally, response to treatment in hematological malignancies is evaluated by light microscopy of bone marrow (BM) smears, but due to more effective therapies more sensitive methods are needed. Today, detection of minimal residual disease (MRD) using immunological and molecular techniques can be 100 times more sensitive than morphology. The main aim of this thesis was to compare and evaluate three currently available MRD methods in childhood acute lymphoblastic leukemia (ALL): (i) real-time quantitative PCR (RQ-PCR) of rearranged antigen receptor genes, (ii) multicolor flow cytometry (FCM) of leukemia-associated immunophenotypes and (iii) real-time quantitative PCR of fusion gene transcripts (RT-PCR).

In paper I, we assessed the applicability of RQ-PCR in a population-based cohort of childhood ALL diagnosed in Sweden between 2002-2006. Clonal IG/TCR rearrangements were identified in the 96% of the 279 ALL cases. Using RQ-PCR, the quantitative range of 10-3 was reached in 93% of B-cell precursor (BCP) ALL and 86% of T-cell ALL (T-ALL) by at least one target gene. In paper II, we compared MRD detection using both RQ-PCR and FCM in the context of NOPHO ALL-2000 protocol. By applying the stratification threshold of ≥0.1% MRD late during induction therapy (day 29), we could demonstrate that both methods can predict the risk of BM relapse but not extramedullary relapse. However, the threshold of ≥0.2% MRD appears to be more optimal using RQ-PCR in BCP ALL, whilst in T-ALL, the results indicate that RQ-PCR is preferable for MRD assessment.

The stability of RNA in vitro is a critical factor when using sensitive molecular techniques such as MRD detection. In paper III, we evaluated the influence on MRD detection when blood is collected in tubes with RNA stabilization reagents (PAX gene Vacutatiner®) compared to collection in EDTA-tubes (non-stabilized). We analyzed 68 matched samples from chronic myeloid leukemia patients and the results indicated that non-stabilized blood processed within 30 hours is preferable for MRD detection.

In paper IV, follow-up samples from eight children with Philadelphia positive (Ph+) ALL were evaluated with the three available MRD methods. MRD measured by the fusion gene transcripts (BCR-ABL1) appeared to be the most sensitive method, however, precise quantification can be difficult and the other methods are thus complementary.

In conclusion, all three applied MRD methods are useful and correlate to each other, although not necessary exchangeable in individual patients. We also conclude that MRD assessment by RQ-PCR, based on rearranged IG/TCR genes and multicolor FCM are predictive for identification of high risk childhood ALL patients.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2009. 64 p.
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine, ISSN 1651-6206 ; 456
Childhood acute lymphoblastic leukemia, minimal residual disease, IG/TCR gene rearrangements, BCR-ABL1 fusion gene transcripts, real-time quantitative PCR, multicolor flow cytometry
National Category
Cell and Molecular Biology
Research subject
urn:nbn:se:uu:diva-101028 (URN)978-91-554-7521-5 (ISBN)
Public defence
2009-05-30, Rudbecksalen, Rudbeck laboratory, Dag Hammarskjölds väg 20, Uppsala, 09:15 (Swedish)
Available from: 2009-05-08 Created: 2009-04-16 Last updated: 2009-05-08Bibliographically approved

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Thörn, Ingrid
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