Acute lymphoblastic leukemia (ALL) is a malignant disease characterized by the uncontrolled proliferation of immature white blood cells in the bone marrow. ALL affects the B-cell (80-85%) or T-cell (20-25%) lineages and is most common in children. While the 5-year event-free survival rate for children with newly diagnosed ALL is over 80 percent, prognosis among children whose cancer recurs is less favorable. If left untreated, ALL ultimately leads to death.
To minimize the risk of an ALL relapse after or during therapy, detection at an early stage is critical to determine if there is a resurgence of cancer cells via minimal residual disease (MRD) monitoring. MRD refers to the proportion of cancer cells among normal bone marrow or blood cells and is regularly used to assess response to treatment at distinct time points.
Standard for MRD Detection
The current gold standard for MRD detection is quantitative singleplex real-time quantitative PCR (qPCR) of clonal immunoglobulin (Ig) and T-cell receptor (TCR) gene rearrangements using allele-specific primers. However, due to clonal tumor evolution or secondary rearrangement processes, there are challenges.
"MRD markers can disappear during treatment, which can lead to false-negative results and poor decision-making in personalized treatments," explained Cornelia Eckert, PhD, from the Department of Pediatric Oncology/Hematology, Charite - Universitatsmedizin Berlin, German Cancer Consortium, and German Cancer Research Center. Therefore, monitoring at least two independent markers per patient is recommended.
Another disadvantage is that "the current gold standard EuroMRD Consortium guidelines call for amplification using singleplex real-time PCR quantification, making testing additional markers more laborious and expensive. They also lead to a higher consumption of patient material," continued Eckert. This might result in a delay in the availability of a sensitive MRD assay for MRD quantification at early follow-up time points during treatment.
New Workflow & Guidelines
To overcome these limitations, researchers from the University of Freiburg, the Charite, and Hahn-Schickard presented a personalized multiplex mediator probe PCR for improved MRD quantification (MRD-MP PCR) in patients with ALL. Their protocol is published in The Journal of Molecular Diagnostics (2022; https://doi.org/10.1016/j.jmoldx.2021.10.001).
In the study, Eckert and co-investigators describe an iterative workflow and guidelines for the successful design of several novel quantitative, multiplex, patient-specific personalized multiplex MP PCRs.
"Multiplexing can significantly improve personalized MRD monitoring of patients, because a higher number of MRD markers per patient can be analyzed at the same time. Even though these patient-specific sequences of cancer cells only differ in a few DNA nucleotides from healthy cells, our multiplex assay can still distinguish between these DNA sequences. Therefore, a broader range of patient-specific sequences can be included in the assay," stated co-investigator Michael Lehnert, PhD, of Hahn-Schickard Freiburg.
The researchers designed four personalized MP PCRs for three MRD markers for ALL simultaneously in one assay (Ig/TCR gene rearrangements, gene fusions, and gene deletions). DNA primer titration was only involved and extended if the assay performance was inefficient in the first step, so that the number of iterations is reduced. Moreover, all assays used target sequence independent, optimized fluorogenic universal reporter molecules, which support quality management and standardization. Additionally, the MRD-MP guidelines enabled software-assisted design that facilitate, accelerate, and standardize the intricate patient-specific assay design for MRD analysis.
To demonstrate the potential transfer of the duplex MP PCR into a routine diagnostic setting, one new duplex assay was applied in a patient case in comparison with the gold standard singleplex test. In this direct comparison, duplex MP PCR was at least as good as the gold standard singleplex hydrolysis probe PCR. The resulting multiplex MP PCRs were able to precisely quantify more MRD markers in less amount of sample material as the gold standard and deliver high quantitative ranges and sensitivities.
"Both fulfill the EuroMRD guidelines and lead to a similar quantitative range and sensitivity for the two detected gene rearrangements," the researchers wrote. Additionally, the authors increased the multiplex level from duplex to 4-plex in a proof of principle and still met the EuroMRD requirements for quantitative and sensitive measurements of MRD.
Conclusions
While their work demonstrates that multiplex MP PCR has the potential to set a new standard in personalized MRD monitoring, the investigators noted that a clinical validation of the assay must be performed in a large representative cohort comparing the multiplex MP approach to the gold standard qPCR approach before it can be used routinely in the clinical environment.
"Cancer is a fatal disease from which not every patient can be cured," Eckert stressed. "After successful clinical validation, patients could benefit from extended MRD monitoring, leading to more precise predictions of therapy response and better patient stratification and outcomes."
First author Elena Kipf, PhD, Hahn-Schickard Freiburg, concluded that "there is a vast variety of DNA marker sequences unique to each leukemia. The MRD-multiplex workflow provides a systematic and reliable way of effective MRD-MP PCR design and optimization and helps the standardization of personal diagnostics." Clinical validation of the test system is currently in progress.
Dibash Kumar Das is a contributing writer.