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Mary Alikian 1,2,*,Olga Tatarinova1, Alistair Reid1, Jane Apperley2,3, Letizia Foroni2,3 1 Imperial Molecular Pathology, Imperial Healthcare Trust, Hammersmith Hospital, London, UK 2 Centre for Haematology, Faculty of Medicine, Imperial College London, London, UK 3 Clinical Haematology, Imperial College Healthcare NHS Trust, London, UK |
Abstract
Tyrosine Kinase Inhibitors (TKIs) are part of the successful clin- ical management of patients with Chronic Myeloid Leukaemia (CML). However, optimal clinical management of CML requires a robust, standardised laboratory assay used at key clinical mile- stones to ensure a successful outcome for patients on TKIs. Quantitative monitoring of %BCR-ABL1IS by reverse transcription quantitative PCR (RT-qPCR) is the gold standard strategy for evalu- ating patient response to therapy and classification into prognostic subgroups. However, it can be challenging to perform in a repro- ducible manner. Reverse-Transcription Digital PCR (RT-dPCR) is an adaptation of this method that could provide the robust and stan- dardised workflow needed for patient stratification.
Recent research from our lab showed that RT-dPCR, using the Europe Against Cancer (EAC) assay, did not significantly improve the sensitivity of residual disease quantification below Major Molecular Response (MMR). The presence of a considerable amount of background noise in the negative samples did not allow for an increase in the sensitivity. However, using RT-dPCR simplified the quantification process eliminating the need for using standard curves and enabling the absolute quantification of the BCR-ABL1 transcript molecules expressed as transcript copies per reaction.
In this study, we validated a new assay, called IDT, for the quan- tification of BCR-ABL1 transcripts on the RainDropTM dPCR platform. The IDT assay uses GUSB as a reference gene and is capable of quantifying the e13a2 and e14a2 transcript types of BCR-ABL1 inde- pendently in each sample in a multiplex format. Using this assay, we quantified MRD in 138 samples from 64 patients enrolled in the UK based De- Escalation and Stopping Treatment of Imatinib, Nilot- inib or sprYcel in Chronic Myeloid Leukaemia (DESTINY) trial and compared the BCR-ABL1 transcripts numbers quantified by both RT- qPCR and RT-dPCR. Although the IDT assay had significantly lower noise levels, the assay did not make a significant difference on the sensitivity compared to RT-qPCR. This result alludes to the fact that samples in deep molecular response are truly disease free.
In this work, we introduce the IDT assay coupled with RT-dPCR on the RainDrop platform for the quantification of BCR-ABL1 trans- cripts for monitoring MRD in CML patients.
http://dx.doi.org/10.1016/j.bdq.2017.02.038
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