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Personalized ddPCR monitoring of rare genetic alterations can help clinicians make timely treatment decisions for patients with leukemia, according to research.

Personalized droplet digital polymerase chain reaction (ddPCR) monitoring of rare genetic alterations can help clinicians make timely treatment decisions for patients with leukemia, according to a study published in Scientific Reports.

“Our study demonstrated the feasibility of applying ddPCR platform in MRD [minimal residual disease] monitoring for patients with high sensitivity,” lead author Beca B. K. Ip, PhD, MSc, and colleagues wrote. “The aim was to design personalized MRD monitoring assays for patients, allowing clinicians to make prompt decision on treatment strategies upon detection of early signs of relapsing disease.”

MRD detection enables clinicians to evaluate treatment response, stratify risk, and predict patient outcomes. Current methods for MRD monitoring, such as quantitative PCR (qPCR), are sensitive but not always available to patients with rare mutations.

The researchers collected bone marrow aspirates or peripheral blood samples from 14 patients with rare genetic translocations, extracted RNA or DNA from each sample, and performed ddPCR assays to detect fusion transcripts or genetic mutations that serve as molecular markers at RNA and DNA levels (atypical BCR::ABL1 e19a2, e23a2ins52, e13a2ins74, rare types of CBFB::MYH11 (G and I), PCM1::JAK2, KMT2A::ELL2, PICALM::MLLT10 fusion transcripts and CEBPA frame-shift and insertion/duplication mutations).

They found that the personalized ddPCR assays benefitted patients by enabling close surveillance at a molecular level during their treatment. For example, one patient carrying an actionable yet atypical BCR::ABL1 fusion transcript (e23a2ins52) maintained a deep molecular response for 4 years after a relapse and timely switch to second-line treatment, and another patient’s donor lymphocyte infusion therapy was guided over more than 2 years.

Kateryna Fedorov, MD, who was not involved in the study, talked with Physician’s Weekly (PW)about how personalized testing for rare genetic alterations impacts the treatment of patients with hematologic malignancies.

PW: What is the most important takeaway from this study?

Dr. Fedorov: Monitoring response to therapy in patients with hematologic malignancies is crucial for personalized care delivery and best outcomes. Positive MRD in acute leukemia indicates higher relapse risk, while achieving deep molecular response in chronic leukemia correlates with better outcomes.

Although most hematologic malignancies harbor genetic mutations or chromosomal alterations, some patients exhibit rare genetic variations that are difficult to identify and monitor with commercially available testing panels. Current methods for MRD monitoring, like qPCR, are sensitive but not readily available for patients with rare genetic alterations.

Personalized testing for rare genetic alterations bridges this gap. The personalized ddPCR assay in this study used custom primers, probes, and thermal cycling conditions for MRD detection and monitoring.

How can ddPCR assays for personalized MRD monitoring affect patient care?

Reliable, personalized ddPCR monitoring of rare genetic alterations could inform treatment decisions for patients who otherwise would have to rely on less sensitive or more generalized, “one-size-fits-all” tests. The advantage of using ddPCR over qPCR or next-generation sequencing is that it allows for increased sensitivity and precision in detecting the genetic alteration. The drawback of ddPCR, as described in the paper, is that it requires the presence of diagnostic samples, customized primers, probes, and assay development.

How feasible is incorporating ddPCR assays for MRD into patient testing?

The materials required for ddPCR testing, including reagents and specialized equipment, are generally available from biotechnology suppliers. However, these materials may be more costly than traditional PCR reagents, which might increase the overall expenditure of testing, especially if not used on a large scale.

Another challenge is the technical expertise needed to perform ddPCR assays. Prior to routine use, laboratory personnel would require specialized training, adding to the complexity and potential costs associated with implementation. Such assays would be limited to specialized laboratories and large academic centers.

In addition, patients’ insurance plans’ coverage of ddPCR testing can vary widely. Challenges in securing coverage may stem from the absence of standardized protocols for ddPCR across multiple laboratories, the need to clinically justify novel MRD targets and validate results for established MRD markers using ddPCR, and insufficient recognition from regulatory and advisory bodies.

What strengths or limitations are worth noting? 

The strength of the study is that it highlights the utility of MRD testing and encourages clinicians to investigate alternative monitoring strategies for rare genetic alterations. Nevertheless, while the scientific community is searching for ever-so-sensitive and specific MRD detection strategies, it is important to remember that prior to clinical use, novel techniques need to be thoroughly tested and validated.

What unanswered questions do you have about ddPCR testing? 

Most patients in this paper had high levels of transcript in the diagnostic sample. It would be interesting to learn more about scenarios where diagnostic samples are unavailable and custom gene fragments must be used to synthesize the fusion transcripts or mutations.  

Is there anything else you would like to add?

The field of hematologic malignancies is rapidly developing, with guidelines being updated frequently. Nevertheless, multiple areas need discovery, innovation, and improvement. It is paramount that clinicians look for novel diagnostic and therapeutic approaches for patients with rare clinical presentations and genetic signatures.