Raising the Bar in MRD Testing

Although cancer is the primary cause of death worldwide, the adoption of minimal residual disease (MRD) testing has made pathologists better-equipped than ever to guide clinical decision-making and improve outcomes via effective, personalized care. Yet even as the role of MRD testing in the clinic expands, molecular pathologists and laboratory medicine professionals still face significant challenges. These include streamlining the MRD testing workflow, navigating a lack of standardized detection approaches, and selecting diagnostic testing technology that is sensitive and accurate enough to optimize the MRD testing process.

After patients undergo curative cancer treatment such as chemotherapy or tumor resection, molecular pathologists evaluate liquid biopsy samples for MRD by quantifying tumor-specific DNA or RNA biomarkers – an indicator of the small number of cancer cells that remain. Though MRD signals are subtle, testing improves cancer care by providing unprecedented insight into prognosis, relapse risk, and recurrence. The practice was initially developed for evaluating and monitoring blood cancers long-term; now, it is increasingly informing solid tumor treatment as well.

Technologies used to assess MRD must be able to detect microscopic signatures of cancer. Although many tactics for cancer diagnosis and monitoring rely on imaging and tissue pathology to evaluate substantial masses, they cannot detect MRD; instead, MRD testing relies heavily on PCR and sequencing approaches. Therefore, pathology labs must consider cost and workload when selecting workflows that use efficient protocols to deliver precise, sensitive results.

Some cancer types – such as chronic myeloid leukemia, metastatic melanoma, and lung, breast, and pancreatic cancers – have well-defined biomarkers with affordable commercial tests, but other types may have mutational profiles unique to each patient. In these cases, molecular profiling of a tumor when it is first discovered lays the foundation for MRD testing; a pathology lab identifies a panel of tumor-specific biomarkers via next-generation sequencing (NGS) for the patient. Subsequently, labs have the option to shift to highly sensitive PCR-based technologies for ongoing monitoring of clinically relevant MRD biomarkers.

Some labs use NGS throughout all stages of the MRD testing workflow, but the most cost-effective and least labor-intensive approaches tend to pair NGS and PCR technologies together to benefit from their complementary strengths. Although PCR-based tools detect fewer targets than NGS, certain platforms are capable of achieving much better sensitivity. Furthermore, PCR assays take minutes instead of hours to set up, cost significantly less per run, and return results within hours instead of days.

Common PCR technologies used for MRD testing include droplet digital PCR (ddPCR), qPCR, and RT-PCR. When selecting one or more of these to incorporate into the testing workflow, consider workload and sensitivity needs. Molecular pathology labs use older mainstay platforms such as qPCR and RT-PCR for a variety of testing, but these require a standard curve for interpretation, which increases the hands-on time for each sample and adds a layer of human error to result interpretation. This makes qPCR and RT-PCR less sensitive than ddPCR and not reliably capable of detecting very low – but clinically relevant – concentrations of MRD.

Interpreting a ddPCR assay does not require a standard curve and therefore offers higher sensitivity and lower technician burden. Instead, prior to amplification in a thermocycler, ddPCR features a step that partitions the sample into about 20,000 droplets, each containing one DNA or RNA molecule. Amplification only occurs in droplets containing the target DNA, causing them to fluoresce and allowing a droplet reader to directly count them to calculate the quantity of target molecules in the original sample.

Pairing ddPCR with an NGS technique represents an ideal combination; NGS can uncover a broad range of potential biomarkers, but has lower sensitivity, making it suboptimal for monitoring low-abundance biomarkers in the blood. ddPCR assays have high sensitivity and precision (capable of counting target molecules as low as 0.001 percent mutant allele frequency), making the NGS-ddPCR combination a better match than either technique alone.

Labs may also employ multi-step testing in their MRD workflows. For example, some labs prefer to identify biomarkers using NGS and conduct initial MRD testing with qPCR or RT-PCR. If the qPCR or RT-PCR results come back negative or inconclusive, they then reflex to a more sensitive ddPCR assay in case low, but clinically relevant, MRD signals are present. Could ddPCR play an increasingly dominant role in MRD testing in future? Given that biomarker researchers use the sensitive, precise, and quantitative nature of ddPCR to establish reference standards and characterize new MRD measurements, I predict that it will.

The growing demand for MRD testing is driving ongoing biomarker investigations and research to uncover new, related applications. Armed with sensitive, efficient testing workflows, molecular pathologists can use MRD data to more closely align clinical decision-making with a patient’s disease progression and recommend treatment sooner upon recurrence. Though best practices are still evolving, we must systematically reconcile common technical and operational challenges that arise in the lab. This is key if we are to maximize pathologists’ ability to advance patient care through an ever-expanding range of MRD assays and ongoing clinical research.