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

Maria Teresa Bochicchio,Daniela Cilloni,Luigiana Luciano,Giovanni Martinelli,Carmen Fava,Santa Errichiello,Daniele Calistri,Giovanna Rege-Cambrin,Emilia Giugliano,Gianantonio Rosti,Giuseppe Saglio,Jessica Petiti,Emanuela Ottaviani,Paola Berchialla,Claudia Venturi,Fabrizio Pane,Filomena Daraio,Barbara Izzo,Enrico M Gottardi

doi:10.3390/cancers13215470

Abstract

Simple SummaryThe introduction to clinical practice of a treatment-free remission approach in chronic myeloid leukemia patients with a stable deep molecular response highlighted how crucial it is to monitor the molecular levels of BCR–ABL1 as accurately and precisely as possible. In this context, the droplet digital PCR (ddPCR) presents an alternative methodology for such quantification. To hypothesize the introduction of this technology in routine practice, we performed a multicentric study that compares ddPCR with the standard methodology currently used. Our results demonstrate that the use of ddPCR in clinical practice is feasible and could be beneficial.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 QXDxTM BCR-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.

Highlights

Chronic myeloid leukemia (CML) is a clonal hematopoietic disorder characterized by increased and unregulated growth of differentiated myeloid cells that accumulate in the blood [1]
A total of 252 measurements were included in this analysis, 126 for the droplet digital PCR (ddPCR) and 126 for the real-time quantitative PCR (RT-qPCR), divided as follows: 60 for group 1, 54 for group 2, 48 for group 3, 60 for group 4, and 30 for group 5
A Student’s t-test did not reveal significant differences between the mean values of BCR–ABL1 obtained with the two techniques (Table 1)

Summary

Introduction

Chronic myeloid leukemia (CML) is a clonal hematopoietic disorder characterized by increased and unregulated growth of differentiated myeloid cells that accumulate in the blood [1]. A qualitative PCR on a peripheral blood sample is mandatory to identify the specific BCR–ABL1 isoform while real-time quantitative polymerase chain reaction (RT-qPCR) in necessary to assess the response to tyrosine kinase inhibitors (TKIs) therapy [3] since the majority of patients taking TKIs obtain a major molecular response (MMR) and about one third to one fourth achieve a deep molecular response (DMR) with fewer detectable leukemic cells [2]. 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

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Conclusion