Exceeding MR5.0 Sensitivity in Routine BCR-ABL1 Analysis Using Multiplex Digital PCR

Objectives and background:

Clinical studies are currently initiated where treatment of CML patients with tyrosine kinase inhibitors is stopped. One of the main criteria for patients to be eligible for these studies is that they must have achieved a molecular response of MR4.0. However, in order to obtain a reliable molecular measurement at the MR4.0 sensitivity level a higher sensitivity is needed in the analysis. We have tested a multiplex droplet digital PCR (ddPCR) approach in order to obtain reliable measurements at the MR5.0 level. When using a standard laboratory protocol with three reference genes and singleplex qPCR, only ¼ of the sample is tested for BCR-ABL1 transcripts. By using multiplex PCR the entire sample can be tested. The advantage of ddPCR is that an absolute concentration is measured and there is no need for calibration or standardisation of the analysis.

Methods:

Blood samples stabilized in Paxgene tubes from 36 CML patients were used for the study. Samples were analysed according to the standard laboratory protocol with qPCR using B2M, BCR and GUSB as reference genes. For the study presented here RNA was purified from 5 million cells using the QiaSymphony RNA kit (Qiagen). 2.5 ug of RNA was used for cDNA synthesis using SuperScript VILO cDNA synthesis kit (LifeTechnologies). Samples were analysed with multiplex dd PCR on the QX100 system (Biorad). The WHO reference genes BCR and GUSB were used as reference. Eight wells were analysed for BCR-ABL1 (FAM labelled probe). Four of the wells were also analysed for BCR and four were analysed for GUSB (HEX labelled probes).

Results:

The median number of GUSB transcripts in the samples was 605,000 copies (range 195,520-934,400). According to the EUTOS “Working definitions of Molecular Response in CML” (ref), MR5.0 sensitivity is obtained when analysing 240,000 GUSB transcripts. All samples except one were above this level. MR5.5 is obtained when analysing 759,000 GUSB transcripts and 20% of the samples (7/36) reached this level. The ddPCR results were compared to the qPCR results and a high concordance was seen in the positive samples (r²=0.94). 21 samples were positive by qPCR, and an additional 4 samples were positive by ddPCR.

Conclusions:

Using the multiplex ddPCR protocol developed 97% of the analysed samples had a sensitivity of MR5.0 or better. Compared to the labs routine qPCR protocol 20% more samples were found positive with ddPCR. Due to the multiplex set-up of the ddPCR the entire cDNA sample can be tested for BCR-ABL1 transcripts. Contrary to qPCR there is no need for calibration of the ddPCR analysis and the method has potential for routine use when a high sensitivity is needed.

References:

“Working definitions of Molecular Response in CML”, EUTOS Molecular Monitoring Steering Group, Nick Cross, Martin Müller, Fabrizio Pane, Andreas Hochhaus, November 2013.