The use of monoclonal gene rearrangement for detection of minimal residual disease in acute lymphoblastic leukemia of childhood

Sykes, Pamela J.; Snell, Lesley; Brisco, Michael J.; Neoh, S. H.; Hughes, Elizabeth; Dolman, Gaynor; Peng, Lihua; Bennett, A.J.; Toogood, Ian; Morley, Alexander A.

doi:10.1038/sj.leu.2400542

Cited by 23 publications

(20 citation statements)

References 4 publications

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“…Simplified techniques using standard PCR, 40,41 RQ-PCR 42 or GeneScan 43,44 have proven to be valid for the study of MRD in ALL. Recently, a simplified and relatively inexpensive technique of MRD detection on day 19 of induction therapy by flow cytometry using a panel of only three monoclonal antibodies was able to identify children with B-lineage ALL with a very good response to treatment.…”

Section: Discussionmentioning

confidence: 99%

A simplified minimal residual disease polymerase chain reaction method at early treatment points can stratify children with acute lymphoblastic leukemia into good and poor outcome groups

Scrideli

Assumpção²,

Ganazza³

et al. 2009

Haematologica

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The online version of this article contains a supplementary appendix. BackgroundMinimal residual disease is an important independent prognostic factor in childhood acute lymphoblastic leukemia. The classical detection methods such as multiparameter flow cytometry and real-time quantitative polymerase chain reaction analysis are expensive, time-consuming and complex, and require considerable technical expertise. Design and MethodsWe analyzed 229 consecutive children with acute lymphoblastic leukemia treated according to the GBTLI-99 protocol at three different Brazilian centers. Minimal residual disease was analyzed in bone marrow samples at diagnosis and on days 14 and 28 by conventional homo/heteroduplex polymerase chain reaction using a simplified approach with consensus primers for IG and TCR gene rearrangements. ResultsAt least one marker was detected by polymerase chain reaction in 96.4% of the patients. By combining the minimal residual disease results obtained on days 14 and 28, three different prognostic groups were identified: minimal residual disease negative on days 14 and 28, positive on day 14/negative on day 28, and positive on both. Five-year event-free survival rates were 85%, 75.6%, and 27.8%, respectively (p<0.0001). The same pattern of stratification held true for the group of intensively treated children. When analyzed in other subgroups of patients such as those at standard and high risk at diagnosis, those with positive B-derived CD10, patients positive for the TEL/AML1 transcript, and patients in morphological remission on a day 28 marrow, the event-free survival rate was found to be significantly lower in patients with positive minimal residual disease on day 28. Multivariate analysis demonstrated that the detection of minimal residual disease on day 28 is the most significant prognostic factor. ConclusionsThis simplified strategy for detection of minimal residual disease was feasible, reproducible, cheaper and simpler when compared with other methods, and allowed powerful discrimination between children with acute lymphoblastic leukemia with a good and poor outcome.

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Section: Discussionmentioning

confidence: 99%

A simplified minimal residual disease polymerase chain reaction method at early treatment points can stratify children with acute lymphoblastic leukemia into good and poor outcome groups

Scrideli

Assumpção²,

Ganazza³

et al. 2009

Haematologica

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“…[17][18][19][20] Sensitivities of around 5% are clinically relevant for initial diagnosis, but they are certainly not sufficient for detection of minimal residual disease (MRD) in ALL patients which requires sensitivities of 10 −4 to 10 −5 . 35 It has been suggested that sensitivities of 10 −2 to 10 −3 as determined by PCR might be predictive for slow remission upon chemotherapy, 36 but such sensitivities cannot easily be reached via heteroduplex analysis of PCR products, unless most of the polyclonal T cells are depleted before DNA or RNA extraction. Nevertheless, heteroduplex PCR analysis of TCR gene rearrangements is sufficiently sensitive for monitoring patients with chronic T cell leukemias as well as patients with oligoclonal T cell proliferations in order to predict the possible outgrowth of a dominant cell population.…”

Section: Figurementioning

confidence: 99%

Heteroduplex PCR analysis of rearranged T cell receptor genes for clonality assessment in suspect T cell proliferations

et al. 1997

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Molecular analysis of T cell receptor (TCR) genes is frequently used to prove or exclude clonality and thereby support the diagnosis of suspect T cell proliferations. PCR techniques are more and more being used for molecular clonality studies. The main disadvantage of the PCR-based detection of clonal TCR gene rearrangements, is the risk of false-positive results due to 'background' amplification of similar rearrangements in polyclonal reactive T lymphocytes. Therefore, PCR-based clonality assessment should include analyses that discern between PCR products derived from monoclonal and polyclonal cell populations. One such method is heteroduplex analysis, in which homo-and heteroduplexes resulting from denaturation (at 94°C) and renaturation (at lower temperatures) of PCR products, are separated in non-denaturing polyacrylamide gels based on their conformation. After denaturation/renaturation, PCR products of clonally rearranged TCR genes give rise to homoduplexes, whereas in case of polyclonal cells heteroduplexes with heterogeneous junctions are formed. We studied heteroduplex PCR analysis of TCR gene rearrangements with respect to the time and temperature of renaturation and the size of the PCR products. Variation in time did not have much influence, but higher renaturation temperatures (Ͼ30°C) clearly showed better duplex formation. Nevertheless, distinction between monoclonal and polyclonal samples was found to be more reliable at a renaturation temperature of 4°C, using relatively short PCR products. To determine the sensitivity of heteroduplex analysis with renaturation at 4°C, (c)DNA of T cell malignancies with proven clonal rearrangements was serially diluted in (c)DNA of polyclonal mononuclear peripheral blood cells and amplified using V and C primers (TCRB genes) or V and J primers (TCRG and TCRD genes). Clonal TCRB and TCRD gene rearrangements could be detected with a sensitivity of at least 5%, whereas the sensitivity for TCRG genes was somewhat lower (10-15%). The latter could be improved by use of V␥ member primers instead of V␥ family primers. We conclude from our results that heteroduplex PCR analysis of TCR gene rearrangements is a simple, rapid and cheap alternative to Southern blot analysis for detection of clonally rearranged TCR genes.

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“…10,15,16 PCR products were separated by polyacrylamide gel electrophoresis and examined visually under UV light after staining with ethidium bromide. Monoclonality (or oligoclonality) was defined by the presence on electrophoresis of one (or several) homogeneous PCR products which were identical in both duplicates.…”

Section: Detection Of Monoclonalitymentioning

confidence: 99%

“…At the one extreme, there are simple techniques, such as detection of a monoclonal gene rearrangement using consensus primers, which is non-quantitative and is capable of detecting leukemia down to a level of approximately 10 −3 . 10,11 At the other extreme, there is detection of a monoclonal gene rearrangement using two clone-specific primers which, together with a sensitive quantitation method such as limiting dilution analysis, 12 is capable of accurate quantitation probably down to 10 −5 to 10 −6 . In between there are several methods of intermediate complexity, in which some specificity for the leukemic clone is obtained by specific probing or use of a single specific primer, and which are more quantitative or less quantitative, depending on the exact detection technique used.…”

Section: Introductionmentioning

confidence: 99%

Comparison of methods for assessment of minimal residual disease in childhood B-lineage acute lymphoblastic leukemia

et al. 2001

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The level of minimal residual disease (MRD) early in treatment of acute lymphoblastic leukemia (ALL) strongly predicts the risk of marrow relapse. As a variety of methods of varying complexity have been separately used for detecting and quantifying MRD, we compared the prognostic utility of three methodsmeasurement of blast percentage on day 14 of treatment, detection of monoclonality on day 14 or day 35, and measurement of MRD by PCR-based limiting dilution analysis on day 14 or day 35. The study group comprised 38 children aged 1-15 with Philadelphia-negative B-lineage ALL who were uniformly treated and followed until relapse or for a minimum of 5 years. We also studied some of the technical factors which influence the ability to detect MRD. Measurement of blast percentage on day 14 by an expert morphologist, detection of monoclonality on day 35, and PCR-based measurement of MRD levels on days 14 and 35 all showed significant ability to divide patients into prognostic groups. Measurement of blast percentage on day 14 by routine morphology or detection of monoclonality on day 14 were not useful. The quality of DNA samples varied greatly, as determined by amplifiability in the PCR. However, virtually all amplifiable leukemic targets in a sample were detectable which suggests that the level of detection achieved by limiting dilution analysis is essentially determined by the amount of DNA which it is practicable to study. We conclude that quantification of MRD at the end of induction provides the full range of prognostic information for marrow relapse but is complex; detection of monoclonality on day 35 is simple and has good positive predictive value; and quantification of MRD on day 14 merits further study. PCR-based methods for measurement of MRD levels should incorporate a correction for variation in DNA amplifiability. Leukemia (2001) 15, 385-390.

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The use of monoclonal gene rearrangement for detection of minimal residual disease in acute lymphoblastic leukemia of childhood

Cited by 23 publications

References 4 publications

A simplified minimal residual disease polymerase chain reaction method at early treatment points can stratify children with acute lymphoblastic leukemia into good and poor outcome groups

A simplified minimal residual disease polymerase chain reaction method at early treatment points can stratify children with acute lymphoblastic leukemia into good and poor outcome groups

Heteroduplex PCR analysis of rearranged T cell receptor genes for clonality assessment in suspect T cell proliferations

Comparison of methods for assessment of minimal residual disease in childhood B-lineage acute lymphoblastic leukemia

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