BCR-ABL1 fusion transcript is the minimal residual disease marker in chronic myeloid leukemia; 2% of patients show unusual breakpoints generating atypical transcripts, not quantifiable by standardized real-time PCR (RT–PCR). Response monitoring is performed by non-quantitative NESTED PCR, useless for evaluating patients’ molecular remission, excluding them from treatment-free-remission protocols. Droplet digital PCR (ddPCR) is highly sensitive technology, allowing an absolute quantification independent of standard curves. Based on this, we have developed assays able to evaluate the molecular response in atypical patients. We designed new ddPCR-based molecular assays able to quantify atypical BCR-ABL1 transcripts, with a detection limit of 0.001%, validated in a cohort of 65 RNA from 11 patients. Fifty samples were identified congruently by ddPCR and NESTED PCR (40 positives and 10 negatives for atypical BCR–ABL1 transcript), while 11 positive samples were detected only by ddPCR. Our results highlight ddPCR usefulness, primarily when the BCR–ABL1/ABL1 level is less than 1.5% and NESTED PCR results are often inaccurate. Furthermore, we identified 3 patients who maintained a deep molecular response for at least one year, who could be considered good candidates for treatment-free remission approaches. Here, we describe a new promising molecular approach, highly sensitive, to monitor atypical BCR–ABL1 patients, paving the foundation to include them in treatment-free remission protocols.

BCR–ABL1 mRNA levels represent the key molecular marker for the evaluation of minimal residual disease (MRD) in chronic myeloid leukemia (CML) patients and real-time quantitative PCR (RT-qPCR) is currently the standard method to monitor it. In the era of tyrosine kinase inhibitors (TKIs) discontinuation, droplet digital PCR (ddPCR) has emerged to provide a more precise detection of MRD. To hypothesize the use of ddPCR in clinical practice, we designed a multicentric study to evaluate the potential value of ddPCR in the diagnostic routine. Thirty-seven RNA samples from CML patients and five from healthy donors were analyzed using both ddPCR QXDx^TMBCR-ABL %IS Kit and LabNet-approved RT-qPCR methodologies in three different Italian laboratories. Our results show that ddPCR has a good agreement with RT-qPCR, but it is more precise to quantify BCR–ABL1 transcript levels. Furthermore, we did not find differences between duplicate or quadruplicate analysis in terms of BCR–ABL1% IS values. Droplet digital PCR could be confidently introduced into the diagnostic routine as a complement to the RT-qPCR.

In recent years, the digital polymerase chain reaction has received increasing interest as it has emerged as a tool to provide more sensitive and accurate detection of minimal residual disease. In order to start the process of data alignment, we assessed the consistency of the BCR-ABL1 quantification results of the analysis of 16 RNA samples at different levels of disease. The results were obtained by two different laboratories that relied on The Qx100/Qx200 Droplet Digital PCR System (Bio-Rad) and Quant Studio 3D dPCR System (Thermofisher) platforms. We assessed the compatibility between the estimated values by linear regression, Bland–Altman bias-plot, and Mann–Whitney nonparametric test. The results confirmed the compatibility of the measures, allowing us tocompute an ‘alignment factor’ (AF), equal to 1.41, which was further validated by a different series of experiments. We conclude that the performed measurements by the two laboratories are comparable, and also equalized through the introduction of an alignment factor.

We evaluated BCR-ABL1 kinetics in patients treated with nilotinib and analyzed whether a dynamic model of changes in BCR-ABL1 levels over time could be used to predict long-term responses.

Molecular detection of the BCR-ABL1 fusion transcripts is necessary for the genetic confirmation of a chronic myeloid leukemia diagnosis and for the risk classification of acute lymphoblastic leukemia. BCR-ABL1 mRNAs are usually identified using a conventional RT-PCR technique according to the BIOMED-1 method. In this study, we evaluated 122 BCR-ABL1-positive samples with the Q-LAMP assay to establish if this technology may represent a valid alternative to the qualitative BIOMED-1 PCR technique usually employed for the detection and the discrimination of the common BCR-ABL1 transcripts (p190 and p210 isoforms). We found a 100% concordance rate between the two methods. Specifically, the p190- and p210-positive samples were amplified by Q-LAMP with a median threshold time (Tt) of 26.70 min (range: 24.45–31.80 min) and 20.26 min (range: 15.25-34.57 min), respectively. A median time of 19.63 was observed in samples displaying both (e13a2/e14a2) p210 isoforms. Moreover, the Q-LAMP assay allowed recognition of the BCR-ABL1 e13a2 and e14a2 isoforms (median Tts 18.48 for e13a2 vs. 26.08 min for e14a2; p < 0.001). Finally, 20 samples harboring rare BCR-ABL1 isoforms (e1a3, e13a3, e14a3, and e19a2) were correctly identified by the Q-LAMP assay. We conclude that the Q-LAMP assay may represent a faster and valid alternative to the qualitative BIOMED-1 RT-PCR for the diagnosis at BCR-ABL1-positive leukemias, especially when samples are analyzed in centers with restricted resources and/or limited technical expertise.

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.