A novel DNA-based test may prove useful for identifying which chronic myeloid leukemia patients with undetectable BCR-ABL1 transcripts can safely discontinue tyrosine kinase inhibitor (TKI) therapy, according to Mary Alikian, Ph.D., of Hammersmith Hospital, London, and her colleagues.
The test can quantify very low levels of residual disease in peripheral blood samples from patients with CML in whom BCR-ABL1 transcripts were undetectable using reverse transcription quantitative polymerase chain reaction (RT-qPCR), the researchers reported in a study published online in the Journal of Molecular Diagnostics.
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Their personalized DNA-based digital PCR method rapidly identifies t(9;22) fusion junctions using targeted next-generation sequencing and generates high-performance DNA-based hydrolysis probe assays that are specific to the unique molecular footprint of each patient’s CML clone. The researchers further enhanced the sensitivity of the DNA-based approach by optimizing the technique for use on a digital PCR (dPCR) platform, which provides absolute molecular quantification without the need for a standard curve. This approach avoids laborious breakpoint mapping and improves sensitivity.
The researchers successfully mapped genomic breakpoints in all samples from 32 patients with early-stage disease. Using DNA-based dPCR, disease was quantified in 46 follow-up samples from 6 of the 32 patients, including 36 samples that were in deep molecular remission.
Digital PCR for BCR-ABL1 DNA detected persistent disease in 81% of the molecular-remission samples, outperforming both RT-dPCR (25%) and DNA-based quantitative PCR (19%), the researchers reported (J Mol Diagn. 2016;18:176e189).
Of CML patients who achieve sustained undetectable BCR-ABL1 transcripts on TKI therapy, about 60% experience the return of detectable disease after stopping TKIs and have to restart treatment. An improved method of identifying patients with the lowest likelihood of relapse would allow safe withdrawal of TKI therapy for the 40% of patients who would remain disease free.
The researchers are currently investigating the impact of residual-disease level as assessed by dPCR at the time of treatment withdrawal on outcome within the UK-based DESTINY clinical trial (Deescalation and Stopping Treatment of Imatinib, Nilotinib or Sprycel in Chronic Myeloid Leukaemia). “If validated in clinical trials of stopping TKI, the technique will permit a more personalized approach to recommendations for dose reduction or drug cessation in individual patients, ensuring that therapy is withdrawn only from patients with the highest likelihood of long-term remission,” they wrote.
Identifying genomic breakpoints as soon as CML is diagnosed would allow for the design and optimization of a patient-specific assay. Patients’ response to therapy would then be monitored via standard RT-qPCR until they have reached molecular response. Thereafter, routine monitoring would be augmented with DNA quantification by dPCR and would benefit from the publication of standardized guidelines, as with RT-qPCR.
In the future, it will therefore be important to explore not only whether the risk of relapse after withdrawal is a feature of the number of residual CML cells but also whether it relates to the degree of transcriptional activity in those cells, the researchers wrote. “We observed that 8% (3 of 36) of the samples were positive by RNA-based but negative by DNA-based methods. Conversely, in samples with detectable BCR-ABL1 DNA, there was heterogeneity in the detectability of transcript by RT-dPCR that appeared to be unrelated to the amount of BCR-ABL1 DNA detected. It should be borne in mind that RT and cDNA synthesis steps remain a potential source of variation affecting cDNA concentration, and therefore these results should be interpreted with caution.”
The researchers had no relevant disclosures. The study was supported by Leading Leukemia Research (LEUKA) charity grant 06/Q0406/47, the National Institute for Health Research Biomedical Research Center Funding Scheme, and the Imperial College High Performance Computing Service.
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