IS THE HIGH-THROUGHPUT DROPLET DIGITAL PCR A NEXT-GENERATION TECHNOLOGY FOR ACCURATE AND SENSITIVE BCR-ABL1 QUANTIFICATION FOR DEEP MOLECULAR RESPONSE MONITORING IN CML?
Author(s): ,
Hana Zizkova
Affiliations:
Institute of Hematology and Blood Transfusion,Prague,Czech Republic
,
Karla Zemanova
Affiliations:
Institute of Hematology and Blood Transfusion,Prague,Czech Republic
,
Hana Klamova
Affiliations:
Institute of Hematology and Blood Transfusion,Prague,Czech Republic
,
Jan Zach
Affiliations:
Institute of Hematology and Blood Transfusion,Prague,Czech Republic
,
Eliska Motlova
Affiliations:
Institute of Hematology and Blood Transfusion,Prague,Czech Republic;University of Chemistry and Technology,Prague,Czech Republic
,
Susanne Saussele
Affiliations:
III. Med. Klinik,Universitätsmedizin Mannheim, der Universität Heidelberg,Mannheim,Germany
,
Francois Xavier Mahon
Affiliations:
Hôpital Haut Lévêque, Centre Hospitalier Universitaire,Bordeaux,France
Katerina Machova Polakova
Affiliations:
Institute of Hematology and Blood Transfusion,Prague,Czech Republic
EHA Library. Zizkova H. 06/12/15; 100479; P227 Disclosure(s)(s): Institute of Hematology and Blood Transfusion
Department of Molecular Genetics
Hana Zizkova
Hana Zizkova
Contributions
Abstract
Abstract: P227

Type: Poster Presentation

Presentation during EHA20: From 12.06.2015 17:15 to 12.06.2015 18:45

Location: Poster area (Hall C)

Background
A sensitive and standardized monitoring of deep molecular response (MR) based on BCR-ABL1 transcript level quantification is an essential part of CML tyrosine kinase inhibitor (TKI) stopping trials. Detailed laboratory recommendations, developed as a part of the European Treatment and Outcome Study (EUTOS) for CML, to enable testing laboratories to score MR in a reproducible manner based on reverse transcription quantitative PCR (RT-qPCR) has been recently published (Cross et al. Leukemia 2015). A new quantification technology droplet digital PCR (ddPCR) is tested in more and more laboratories with promising high quantification accuracy and sensitivity.

Aims
To assess differences in accuracy and sensitivity of BCR-ABL1 transcript levels quantification using ddPCR and standardized RT-qPCR. In addition to that to compare the levels of BCR-ABL1 at mRNA and DNA levels measured by ddPCR.

Methods

The RT-qPCR used in our EUTOS MR4.5 laboratory is standardized having regularly validated conversion factors for data reporting in the international scale (IS) using ABL1 or GUSB control genes. The system QX-200 (Biorad) was used for ddPCR. The same RT-qPCR primers and probes were applied for ddPCR. Unlike to singleplex RT-qPCR, TaqMan probes were differently labelled for multiplex ddPCR. Using each method all samples were analyzed in doublets.



Results

The WHO certified ERM-AD623 standards (n=6) were used to compare the quantification accuracy of both methods. Obtained gene copies ratios (BCR-ABL1/ABL1 and BCR-ABL1/GUSB) in the standards by singleplex RT-qPCR were in range 0.63-1.11 (Me=0.85). Singleplex ddPCR determined ratios in range 0.92-1.29 (Me=1.02). Multiplex ddPCR improved quantification accuracy with ratios 0.93-1.0 (Me=0.99).

Samples of 31 CML patients treated with TKIs with BCR-ABL1 mRNA levels ranging 0.01-10% IS were used for data comparison. The Bland-Altman bias plot was used to determine bias between RT-qPCR and ddPCR showing antilog bias 0.54-fold in 31 paired results.

To compare BCR-ABL1 detection sensitivity of both methods we tested 66 samples from the time of MR4.0 confirmation and after TKI discontinuation of 7 patients enrolled in the EURO-SKI study (Europe Stops TKI in CML). The total number of paired results from RT-qPCR vs. ddPCR of samples with BCR-ABL1 mRNA levels below 0.01% IS was 37 with determined antilog bias 0.23-fold. The number of BCR-ABL1 negative samples by RT-qPCR vs. ddPCR was 26/66 in MR4.5-MR5.0 sensitivity vs. 6/66, respectively. Moreover, 26 samples of 4/7 EURO-SKI patients were assessed by ddPCR for the levels of genomic BCR-ABL1 fusions using patient-specific assays. We found significant correlation between DNA and mRNA BCR-ABL1 levels (r=0.9038, P<0.0001).



Summary
We found a higher quantification accuracy of ddPCR in contrast to RT-qPCR for which standard calibration curves need to be performed to mathematically calculate cDNA copy numbers in unknown samples. This probably represents an important variable. We found comparable results of both methods for BCR-ABL1 mRNA quantification within the range 0.01-10% IS. Approximately a log higher BCR-ABL1 mRNA levels in EURO-SKI MR4.0-MR5.0 samples were detected by ddPCR in comparison to RT-qPCR, assuming that higher sensitivity of ddPCR may be important for TKI stopping treatment management. Interestingly, BCR-ABL1 DNA and mRNA levels measured by ddPCR correlated significantly. A larger cohort of samples needs to be analyzed to elucidate the clinical importance of DNA measurement.

Supported by MZCR project no. 00023736 and EURO-SKI research consortium



Keyword(s): BCR-ABL, Chronic myeloid leukemia, Gene expression, Tyrosine kinase inhibitor

Session topic: Chronic myeloid leukemia - Biology

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