Researchers have developed a new protocol for monitoring acute lymphoblastic leukemia (ALL) to inform more effective treatment strategies and detect disease recurrence, such as cancer recurrence.

The personalized mediator probe PCR (MP PCR) uses multiple genomic cancer cell markers in a single assay, and improves monitoring clonal tumor evolution to detect a relapse sooner and avoid false negative results. The protocol is detailed in The Journal of Molecular Diagnostics, published by Elsevier.

The survival rate for children with ALL—the most common cancer in children—has increased over 80% over the last several decades. However, the prognosis for children with cancer recurrence remains unfavorable. Therefore, minimal residual disease (MRD) monitoring is an important prognostic factor for treatment response and patient stratification. MRD monitoring uses highly sensitive real-time PCR to measure the amount of cancer cells among normal cells.

“MRD markers can disappear during treatment, which can lead to false-negative results and poor decision-making in personalized treatments,” says Principal investigator Cornelia Eckert, PhD, Department of Pediatric Oncology/Hematology, Charité – Universitätsmedizin Berlin, German Cancer Consortium (DKTK) and German Cancer Research Center (DKFZ). Consequently, monitoring at least two independent markers per patient is recommended.  “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.”

To overcome current limitations, Eckert and co-investigators established the personalized MP PCR, an iterative workflow and guidelines for designing multiplex real-time PCRs to monitor up to four MRD markers for ALL simultaneously in one assay. When tested on DNA in bone marrow samples from patients with ALL, the MP PCRs met the EuroMRD gold standard guidelines and level of sensitivity for clinical decision-making.

“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,” says co-investigator Michael Lehnert, PhD, Hahn-Schickard Freiburg. “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.”

In order to deal with challenges inherent to multiplex PCR, the researchers developed an efficient iterative workflow for PCR design and optimization. DNA primer titration is only involved and extended if the assay performance is not sufficient in the first step, so that the number of iterations is minimized. 

“There is a vast variety of DNA marker sequences unique to each leukemia,” says first author Elena Kipf, PhD, Hahn-Schickard Freiburg. “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.”

While multiplex MP PCR has the potential to set a new standard in personalized MRD monitoring, the researchers note it must be clinically validated in a representative cohort of ALL patients.

Featured image: MP PCR-design and -analysis can be done with different software tools already available. In addition, specialized software for multiplex MRD-PCR-design (Assay Manager GNWI mbH, Germany) and standardized analysis (ValidScale Hahn-Schickard e.V., Germany) have been under clinical validation. Photo: The Journal of Molecular Diagnostics