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Digital PCR Approach Can Detect CML Present in Patients at Low Levels, Study Says

NEW YORK (GenomeWeb) – Digital PCR can better detect hints of residual disease in chronic myeloid leukemia patients than other PCR-based approaches, according to a team of UK researchers.

With tyrosine kinase inhibitor (TKI) treatment, CML can be rendered nearly undetectable in patients. But while some patients remain disease-free after treatment is halted, others can relapse. This, the researchers said, highlights the need for ways to detect even low levels of disease.

As they reported today in the Journal of Molecular Diagnostics, the team led by Imperial College London's Alistair Reid developed an approach that combines next-generation sequencing to identify the BCR-ABL1 fusion oncogene that is the hallmark of CML with DNA-based digital PCR to then quantify its presence in patients. Reid and his colleagues further tested this approach in comparison with other PCR-based methods in three dozen samples from six patients, and found that dPCR could detect disease when the others could not.

"If validated in clinical trials of stopping TKIs, this 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approach to recommendations for dose reduction or drug cessation in individual patients, ensuring that therapy is withdrawn only from patients with the highest chance of long-term remission," co-author Jane Apperley, also from Imperial College, said in a statement.

For their approach, Apperley, Reid, and their colleagues used next-generation sequencing to identify breakpoint junctions in 32 CML patients being treated at Hammersmith Hospital. All the junctions mapped by next-gen sequencing in the 32 patients were confirmed by Sanger sequencing.

For each of the breakpoints identified, the researchers designed primers to amplify across the fusion junction and generated high-performance DNA-based hydrolysis probe assays for each patient's particular CML clone. They then optimized it for use on a dPCR platform.

"The technique we describe, with which we successfully mapped a disease-specific junction in all patients tested, is relatively simple, cost effective, and suited to a high-throughput laboratory," Apperley said.

Currently, CML disease level is typically gauged by RT-qPCR.  But, as the researchers noted, some 60 percent of patients whose BCR-ABL1 fusions aren't detectable by RT-qPCR experience disease recurrence when TKI treatment is stopped.

The researchers compared how well RT-qPCR, qPCR, dPCR, and RT-dPCR fared in detecting low levels of BCR-ABL1-positive disease in 36 samples from six patients and 10 positive control samples that had detectable BCR-ABL1 fusions.

Without a preamplification step, dPCR saw residual disease in 16 of the 36 samples. When the researchers added a preamplification step, that number rose to 29 of the samples, or 80.5 percent. At the same time, RT-dPCR found nine of the 36 samples to have detectable disease. That is, 23 of the 27 samples judged to be negative by RT-dPCR were found to be positive via dPCR. Both approaches found detectable disease in all 10 control samples.

Meanwhile, qPCR uncovered disease in seven of the 36 samples; dPCR also uncovered disease in these samples, indicating that qPCR was unable to detect positivity in three-quarters of the dPCR-positive samples. In other words, dPCR detected persistent disease in nearly 81 percent of seemingly molecular-remission samples, while RT-dPCR detected it in 25 percent and DNA-based qPCR in 19 percent of samples.

"We conclude that dPCR for BCR-ABL1 DNA is the most sensitive available method of residual disease detection in CML and may prove useful in the management of TKI withdrawal," Apperley said.

She and her colleagues noted in their paper that they are currently looking into how assessing residual-disease level using dPCR at the time of treatment withdrawal affects outcomes as part of the De-escalation and Stopping Treatment of Imatinib, Nilotinib, or Sprycel in Chronic Myeloid Leukemia (DESTINY) clinical trial. If validated, this would enable a more personalized approach to reducing or stopping drug treatments, the researchers added.