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23 Sep 2021 - 25 Sep 2021

Molecular Assays Compared For MRD Detection In B-ALL

By LabMedica International staff writers
Posted on 11 Aug 2021
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The SuperScript III Reverse Transcriptase (RT) kit provides increased specificity with gene-specific primers (GSPs) and the highest cDNA yield of all RTs (Photo courtesy of Thermo Fisher Scientific).
The SuperScript III Reverse Transcriptase (RT) kit provides increased specificity with gene-specific primers (GSPs) and the highest cDNA yield of all RTs (Photo courtesy of Thermo Fisher Scientific).
B-cell precursor acute lymphoblastic leukemia (B-ALL) is the most common malignancy in children, and it may be associated with gene alterations that regulate B-cell development. The prognostic significance of minimal residual disease (MRD) in children with acute lymphoblastic leukemia (ALL) has been demonstrated.

Two quantitative polymerase chain reaction (qPCR) based-methods, for clonal immunoglobulin or T-cell receptor gene (Ig/TCR) rearrangements and for fusion transcripts, are widely used for the measurement of minimal residual disease (MRD) in patients with B-precursor acute lymphoblastic leukemia (B-ALL).

A large team of Hematology Oncologists at Chang-Gung Memorial Hospital (Taipei, Taiwan) and their colleagues collected bone marrow samples at diagnosis and at different time points after therapy from patients with B-ALL. The diagnostic bone marrow (BM) mononuclear cells (MNC) were immediately enriched by Ficoll-Hypaque density gradient centrifugation, subjected to RNA extraction by using Trizol reagent (Invitrogen Corporation, Carlsbad, CA, USA), and then followed by using RT-PCR assays to detect the major fusion transcripts for diagnostic work up and risk stratification.

The MRD of bone marrow samples from 165 patients carrying the three major fusion transcripts including 74 BCR-ABL1, 54 ETV6-RUNX1, and 37 TCF3-PBX1 were analyzed by using the two qPCR-based methods. Complementary DNA (cDNA) was synthesized by using the Invitrogen Superscript II Rnase H2 reverse transcriptase kit with random hexamer. RT-qPCR with TaqMan assay for the measurement of the three major fusion transcripts was performed with ABL1 gene as internal control on the ABI 7700 (before 2009) or 7900 (after 2009) (Applied Biosystems, Foster City, CA, USA). BIOMED-2 consensus primers for heteroduplex analysis were used to amplify the Ig or TCR locus on gDNA.

The coefficient correlation of both methods was good for TCF3-PBX1, and BCR-ABL1 (ALL and moderate for ETV6-RUNX1). The concordance was perfect for TCF3-PBX1 ALL (97.2%), substantially concordant for ETV6-RUNX1 ALL (87.1%), and only moderate for BCR-ABL1 ALL (70.6%). The discordant MRD, positive for only one method with a difference greater than one log, was found in four of 93 samples (4.3%) with ETV6-RUNX1, 31 of 245 samples (12.7%) with BCR-ABL1, and none of TCF3-PBX1 ALL. None of the eight non-transplanted patients with BCR-ABL1-MRD (+)/Ig/TCR-MRD (-) with a median follow-up time of 73.5 months had hematologic relapses.

The authors concluded that their results demonstrated that each fusion transcript had a trend of specific VH usage. Excellent MRD concordance between the two qPCR-based methods in TCF3-PBX1 ALL indicated that RT-qPCR is the most cost-laboriousness-effective method for TCF3-PBX1 ALL whereas Ig/TCR-qPCR is a more reliable method to guide MRD-adapted therapy in BCR-ABL1 ALL. The study was published on July 25, 2021 in The Journal of Molecular Diagnostics.

Related Links:

Chang-Gung Memorial Hospital
Thermo Fisher


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