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Minimal residual disease assessment in childhood acute lymphoblastic leukemia: Results of a Swedish multi-centre study comparing real-time PCR and multi-colour flow cytometry
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.
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology, Hematology and Immunology. (Molecular Hematology)
Division of Hematology & Transfusion Medicine, Department of Laboratory Medicine, Lund University, Lund, Sweden.
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2009 (English)Other (Other academic)
Abstract [en]

In this Swedish multi-center study of early treatment response in childhood acute lymphoblastic leukemia (ALL), we evaluated the concordance between multicolour flow cytometry (FCM) and real-time quantitative polymerase chain reaction (RQ-PCR) for assessment of minimal residual disease (MRD). Multiple time points (i.e. day 15, 29, 50 and 106) were evaluated with the NOPHO (Nordic Society of Pediatric Hematology and Oncology) ALL 2000 treatment protocol as backbone. During 2002-2006, 334 children were diagnosed with ALL, where 228 had paired samples taken at any of the four time points. With the detection level of 0.1%, the concordance between RQ-PCR and FCM was 90% in the 726 paired samples analyzed. At day 29, the correlation between the methods was greater with MRD levels >0.1% (rs=0.7, p<0.001) than below (rs=0.2, p=0.024). MRD levels higher than 0.1% at day 29 was a significant predictor of higher risk of having a bone marrow relapse. This was true both for BCP ALL and T-ALL analysed with either FCM or RQ-PCR, although RQ-PCR was a better discriminator than FCM in T-ALL. However, using the NOPHO ALL 2000 protocol, our data indicate that a higher cut-off value (0.2%) should be applied in BCP ALL when using RQ-PCR as MRD method. In contrast, MRD levels ≥ 0.1%, analysed with either method late during induction therapy, was not a predictor of isolated extramedullary relapse. We therefore conclude that MRD assessment by RQ-PCR based IG/TCR rearrangement and multicolour FCM monitoring can be used as a clinical tool if the aim is to find childhood ALL cases with increased risk of having bone marrow relapses.

Place, publisher, year, pages
2009.
Keyword [en]
minimal residual disease, childhood acute lymphoblastic leukemia, flow cytometry, RQ-PCR, rearranged IG/TCR genes
National Category
Cell and Molecular Biology
Research subject
Pathology
Identifiers
URN: urn:nbn:se:uu:diva-101003OAI: oai:DiVA.org:uu-101003DiVA: diva2:211592
Available from: 2009-04-16 Created: 2009-04-16 Last updated: 2010-11-05Bibliographically approved
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.
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine, ISSN 1651-6206 ; 456
Series
Keyword
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
Pathology
Identifiers
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)
Opponent
Supervisors
Available from: 2009-05-08 Created: 2009-04-16 Last updated: 2009-05-08Bibliographically approved

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