RT-qPCR and RT-Digital PCR: A Comparison of Different Platforms for the Evaluation of Residual Disease in Chronic Myeloid Leukemia

Abstract

BACKGROUND Tyrosine kinase inhibitors (TKIs) are the cornerstone of successful clinical management of patients with chronic myeloid leukemia (CML). Quantitative monitoring of the percentage of the fusion transcript BCR-ABL1 (breakpoint cluster region–c-abl oncogene 1, non-receptor tyrosine kinase) BCR-ABL1IS (%BCR-ABL1IS) by reverse transcription–quantitative PCR (RT-qPCR) is the gold standard strategy for evaluating patient response to TKIs and classification into prognostic subgroups. However, this approach can be challenging to perform in a reproducible manner. Reverse-transcription digital PCR (RT-dPCR) is an adaptation of this method that could provide the robust and standardized workflow needed for truly standardized patient stratification. METHODS BCR-ABL1 and ABL1 transcript copy numbers were quantified in a total of 102 samples; 70 CML patients undergoing TKI therapy and 32 non-CML individuals. 3 commercially available digital PCR platforms (QS3D, QX200 and Raindrop) were compared with the platform routinely used in the clinic for RT-qPCR using the EAC (Europe Against Cancer) assay. RESULTS Measurements on all instruments correlated well when the %BCR-ABL1IS was ≥0.1%. In patients with residual disease below this level, greater variations were measured both within and between instruments limiting comparable performance to a 4 log dynamic range. CONCLUSIONS RT-dPCR was able to quantify low-level BCR-ABL1 transcript copies but was unable to improve sensitivity below the level of detection achieved by RT-qPCR. However, RT-dPCR was able to perform these sensitive measurements without use of a calibration curve. Adaptions to the protocol to increase the amount of RNA measured are likely to be necessary to improve the analytical sensitivity of BCR-ABL testing on a dPCR platform.

Digital PCR improves the quantitation of DMR and the selection of CML candidates to TKIs discontinuation

“…In the last years, the digital PCR (dPCR) has emerged as a more sensitive and accurate detection tool of minimal residual disease (MRD) and this increased the interest for its use in the clinical practice. [20][21][22] The dPCR provides an absolute target sequence quantity, and recently, the alignment of the dPCR values <0.468 BCR-ABL1 copies/µL (as we previously described) showed a longer DMR duration (P = 0.0220) and mainly belonged to MR 4.5-5.0 (P = 0.0442) classes compared to patients with higher dPCR values. Among the 142 patients, 111 (78%) discontinued the TKI treatment; among the 111 patients, 24 (22%) lost the MR 3.0 or MR 4.0 .…”

“…In order to routinely apply dPCR for BCR‐ABL1 detection and quantification for a personalized management of CML patients, other efforts have to be performed in the standardization of the procedures and in the alignment of the results generated by the different dPCR platforms …”

“…In the last years, the digital PCR (dPCR) has emerged as a more sensitive and accurate detection tool of minimal residual disease (MRD) and this increased the interest for its use in the clinical practice . The dPCR provides an absolute target sequence quantity, and recently, the alignment of the methods for BCR‐ABL1 transcript quantification by using the Qx100/Qx200 Droplet Digital PCR System (Biorad) and the QuantStudio 3D Digital PCR System (Thermofisher) has been accomplished…”

Molecular detection of minimal residual disease in multiple myeloma

“…However, standardized criteria for data interpretation have not yet been established, and the definition of positivity and PNQ vary substantially in different studies (Drandi et al , ; Della Starza et al , ). Of note, Alikian et al () compared the performance of RQ‐PCR with three dPCR platforms, i.e. QuantStudio 3D (Thermo Fisher), QX200 (Bio‐Rad) and RainDrop (RainDance), showing considerable false positivity rates in the no‐template control in the QuantStudio 3D and RainDrop, but not in the QX200 platform.…”

Quantitative detection of IKZF1 deletion by digital PCR in patients with acute lymphoblastic leukemia

“…9 Digital polymerase chain reaction (PCR) is an alternative PCR technology that allows accurate quantitation of the total copy number of initial targets present in a sample without the need for traditional standard samples. 10,11 The principle of digital PCR (dPCR) is based on dilution and partitioning of a sample into hundreds to millions of separate reaction units, so that each partition contains, statistically, a single copy or no copies of the target of interest. By counting the number of "positive" partitions vs "negative" partitions, the total copy number of a DNA molecule in the original sample can be determined exactly.…”