Standardization of BCR-ABL1 p210 Monitoring: From Nested to Digital PCR

Abstract

The introduction of tyrosine kinase inhibitors in 2001 as a targeted anticancer therapy has significantly improved the quality of life and survival of patients with chronic myeloid leukemia. At the same time, with the introduction of tyrosine kinase inhibitors, the need for precise monitoring of the molecular response to therapy has emerged. Starting with a qualitative polymerase chain reaction, followed by the introduction of a quantitative polymerase chain reaction to determine the exact quantity of the transcript of interest-p210 BCR-ABL1, molecular monitoring in patients with chronic myeloid leukemia was internationally standardized. This enabled precise monitoring of the therapeutic response, unification of therapeutic protocols, and comparison of results between different laboratories. This review aims to summarize the steps in the diagnosis and molecular monitoring of p210 BCR-ABL1, as well as to consider the possible future application of a more sophisticated method such as digital polymerase chain reaction.

Droplet Digital PCR for BCR–ABL1 Monitoring in Diagnostic Routine: Ready to Start?

“…Although we were able to demonstrate that ddPCR has a good agreement with RT-qPCR, ddPCR resulted in greater precision (higher repeatability) than RT-qPCR for all the levels of disease. Moreover, the ddPCR was found to be more reproducible than RT-qPCR in all the disease levels, meeting the efforts made by the CML community to standardize the results [5,7,15]. The improved reproducibility and repeatability of ddPCR compared to RT-qPCR which we observed, may partly explain the reason for the superiority of ddPCR in predicting relapses in TFR patients [14,[21][22][23][24][25].…”

“…In addition to the EUropean Treatment Outcome Study (EUTOS) consortium, the Italian laboratory network LabNet (i) is actively involved in the standardization of mBCR-ABL1 quantification and (ii) properly coordinates quality control rounds in order to monitor and eventually improve the network laboratories' performances. The first point has been improved over the years thanks to the introduction of the use of a laboratory-specific conversion factor [7]. Furthermore, in an era in which DMR is becoming the target to be achieved in common clinical practice and represents the mandatory prerequisite for a treatment-free remission approach [3], a highly sensitive and accurate technique for BCR-ABL1 levels monitoring is necessary.…”

Alignment of Qx100/Qx200 Droplet Digital (Bio-Rad) and QuantStudio 3D (Thermofisher) Digital PCR for Quantification of BCR-ABL1 in Ph+ Chronic Myeloid Leukemia

“…So far, RT-qPCR is considered a gold standard for monitoring molecular response and diagnosis of CML patients. Its process of international standardization has been active for more than 20 years, going through all the stages of results validation, conversion factor implementation, and expressing the results on an international scale, as well as creating reference material for the proper quantification of BCR-ABL1 transcripts [4,20,[23][24][25][26][27]. Despitemany efforts to standardize the method, RT-qPCR still carries considerable uncertainty, especially in detecting very low levels of the disease [28].…”

Revealing the Mysteries of Acute Myeloid Leukemia: From Quantitative PCR through Next-Generation Sequencing and Systemic Metabolomic Profiling

“…The technical similarities and differences between dPCR and RT-qPCR are well defined, and the most important ones are listed as follows: (i) dPCR consists of thousands of individual PCR reactions divided during analysis while in RT-qPCR there is only one exponential amplification of the nucleic acids; (ii) dPCR measures the absolute number of the molecule of interest, while RT-qPCR determines the relative number using a standard curve; and (iii) dPCR has higher sensitivity and better accuracy compared to RT-qPCR [38,39]. The attractiveness of dPCR also increases with the fact that it does not require the use of standard reference curves.…”