Approximately 10 million people died from cancer worldwide in 2020, and 19.3 million individuals received new cancer diagnoses.1 Furthermore, by cancer type, the incidence of hematological malignancies per year (such as leukemia, lymphoma, and myeloma) is outpacing that of solid tumors, with the number of new leukemia cases in the United States doubling in the last decade.2 Overall, experts expect that the number of cancer cases will continue to grow as populations reach unprecedentedly long lifespans and experience increased exposure to risk factors like sedentary lifestyles, smoking, poor diet, and excessive body weight. Based on these factors, they predict 28.4 million new cancer cases in 2040.1

To address this expected rise, the medical community is honing its assessment and treatment practices to improve outcomes for affected individuals. Among these initiatives is an increased emphasis on measuring the small number of cancer cells—measurable residual disease (MRD)—that remain after curative treatment and post-adjuvant therapy. These measurements help clinicians predict a patient’s risk of relapse and enable them to intervene earlier in cases of recurrence.

While imaging techniques and cell sorting have long been the gold standard for diagnosing and monitoring cancer, their ability to accurately measure MRD is lacking. Instead, labs run various molecular assays to detect and quantify tumor cell biomarkers circulating in the blood as a means of testing for MRD. Mounting data demonstrate that ultra-sensitive detection methods like droplet digital PCR (ddPCR) can provide critical insight into a patient’s progress throughout treatment, augmenting MRD testing’s role in the oncology clinic.

The Value of MRD Testing

After a patient has gone through the gamut of initial diagnostic tests to inform treatment decisions, MRD testing can provide doctors with ongoing guidance on how the patient’s long-term care should proceed. Any remaining cancer cells in the body can become active and start to multiply, causing relapse. Therefore, determining if a patient is MRD-positive as early as possible can be crucial for modulating the treatment plan.

As the term measurable residual disease suggests, the cancer cells remaining after curative treatment may be so rare that they do not cause immediate physical signs or symptoms. Only molecular testing is sensitive enough to detect MRD, raising the potential to detect disease recurrence weeks to months earlier than imaging techniques. Molecular methods to measure MRD in patients, such as liquid biopsy, also come with significant benefits—in contrast to tissue biopsies, they are noninvasive and tend to be less expensive. Clinicians need only a basic blood draw to perform a liquid biopsy to measure tumor-specific biomarkers, usually nucleic acid mutations within circulating tumor DNA (ctDNA).

Patients with symptoms of cancer first undergo diagnostic procedures to image and molecularly characterize their tumor, and this lab work establishes the tumor-specific biomarkers for MRD testing early on. Patients may initially receive neoadjuvant therapy to reduce tumor size before receiving a curative treatment such as bone marrow transplant for leukemia or tumor resection. At this stage, clinicians and laboratory professionals may measure biomarkers of ctDNA to establish an early prognosis. After completing a curative treatment plan, a clinician will measure MRD to gain a broader understand of a patient’s prognosis and assess risk. In high-risk cases, this testing can indicate the need for post-adjuvant therapy, while in low-risk cases, it may trigger cessation of treatment in favor of long-term monitoring.3, 4

Playing to the Strengths of Current MRD testing Technologies

As the medical field continues to work toward relating MRD testing to clinical outcomes, the promise of MRD has led to rising demand for technologies that can quickly deliver accurate and reproducible results. As a result, scientists have turned to molecular technologies to make the most of this oncology approach.

Common molecular platforms include PCR-based DNA and RNA testing methods like quantitative PCR (qPCR) and quantitative reverse transcriptase PCR (RT-PCR). qPCR and RT-qPCR are often the default in the clinic because medical labs have used these technologies for years. Furthermore, the instruments are accessible and workers are familiar with the equipment protocols. However, meaningful MRD measurements require high sensitivity, and qPCR and RT-PCR methods typically have a detection limit of 0.1-1.0% of target gene species—not sensitive enough to detect low levels of MRD.3 Standardization is also an issue, since qPCR and RT-PCR require standard curves to interpret semi-quantitative results. Researchers using these tools may only compare MRD test results across multiple laboratories after painstakingly validating assays and establishing appropriate controls.

An alternative is ddPCR, a method that can directly count the number of target molecules in a sample without the need for standard curves. This technology divides the sample into approximately 20,000 droplets containing one or a few DNA fragments. The droplets with the target DNA are amplified in a thermocycler and analyzed by a droplet reader that counts the positive droplets based on the fluorescent signal they emit. Lab workers can calculate the precise number of target DNA molecules down to a limit of detection of 0.1% using the number of positive droplets.3,5 The ddPCR method has gained popularity in recent years because it involves a simple workflow and produces sensitive and accurate results beyond those of standard qPCR and RT-PCR.

Some labs use next-generation sequencing (NGS) to complement ddPCR for highly sensitive molecular detection throughout oncology workflows. Technicians use NGS technology to sequence millions of DNA fragments in a massively parallel reaction, aligning them to a reference sequence to pinpoint mutations. Targeted and untargeted NGS techniques may capture many biomarkers simultaneously.6 However, this technique is less sensitive and more expensive than ddPCR. It can also take weeks to return results, especially if a medical lab must send samples to a central facility. Thus, workflows that use NGS and ddPCR most effectively play to their strengths. NGS is indispensable for identifying a panel of tumor-specific mutations, whereas ddPCR can be used with serial liquid biopsies to quickly and precisely track select biomarkers over time. These combined techniques generate data that offer more significant clinical value than either standalone system.

The Future of MRD Testing

Although MRD testing can provide substantial insight, challenges still limit clinical adoption. Budget and staffing capabilities are limiting factors for any medical lab, so reduced testing costs, multiplexing capabilities, and a standardized and secure way to design assays for individual patients will be vital to accelerating the adoption process for this approach.

For every patient, MRD tests typically must assess one or a few biomarkers specific to an individual’s cancer. Therefore, developing a more comprehensive array of commercial, turnkey tests could help lower testing costs. We already see the utility of some inexpensive, commercial tests for cancers with well-defined biomarkers. But for other cancers, labs must still use NGS to establish personalized biomarkers before developing personalized MRD tests—a relatively costly process. This challenge is especially true of MRD testing in solid tumor cancers, which has historically lagged behind MRD testing for blood cancers because solid tumors tend to shed lower amounts of ctDNA.

Currently, the approach to identifying biomarkers, collecting samples, and measuring MRD is still not standardized for solid tumors. However, the field may become more solidified as lab techniques improve; namely, unified sampling practices to maximize DNA concentration and multiplexed approaches to viewing more biomarkers simultaneously with high sensitivity.

In addition, viewing more biomarkers at once could help improve care and reduce costs for all cancer patients. If clinicians could correlate concrete molecular biomarkers like MRD levels with softer biomarkers like fever and blood cell count, they might be able to establish indicators suggesting the patient is at risk of recurrence and in need of an MRD test.

Conclusion

The future of MRD testing looks promising, as ultra-sensitive MRD testing methods have already proven useful in numerous clinical studies. As research efforts continue toward standardization and more widespread adoption of the available techniques, researchers will uncover new uses and expand MRD testing strategies. As medical advances continue to drive the field’s evolution, the momentum will stimulate increased awareness amongst the clinical community and patients about MRD testing, further unifying practices and leading to better patient outcomes.

Lucia Cavelier, PhD, is an associate professor at Karolinska Institute and works as a clinical laboratory geneticist at Clinical Genetics, Karolinska University Hospital with a focus on diagnostics in hematology. She got her PhD in Medical Genetics at Uppsala University and performed postdoctoral studies at the Lawrence Berkeley National Laboratory. Lucia has worked for many years as clinical laboratory geneticist at Uppsala University Hospital and she was the director of the clinical genomics facility at Uppsala University, SciLifeLab. She has been a leading voice for the implementation of NGS and novel technologies in the clinical laboratory environment.

Tatjana Pandzic, PhD, obtained her PhD in cancer genetics at Karolinska Institute and performed her postdoctoral studies at Stockholm University and Uppsala University. Since 2016 she has served as a clinical laboratory geneticist in the clinical genetics lab at Uppsala University Hospital with a focus on molecular diagnostics in hematology. Tatjana has been involved in the implementation of NGS and ddPCR and is also in charge of R&D for hematology at SciLifeLab, Uppsala University.

Case Study 1

Using MRD Testing to Redefine the Treatment of Cancers

Several studies have highlighted the advantages of enhanced sensitivity for MRD testing. For example, treating chronic myeloid leukemia (CML) with tyrosine kinase inhibitors (TKIs) can often be remarkably effective, expanding life expectancy in patients from about 6 years to a full lifespan.7 However, a patient’s quality of life can diminish due to side effects from the TKI treatment. To address this issue, the extensive, US-based LAST Study used MRD monitoring to track the CML biomarker BCR-ABL1 and establish metrics that could help identify patients at low risk of recurrence who could safely stop TKI therapy.8 The prospective study assessed 172 patients who had been on TKI therapy for 3 or more years. The patients achieved substantial molecular response as a result of treatment, defined as BCR::ABL1 transcript levels below 0.01%. Their BCR::ABL1 transcript level remained at or below this level for 2 or more years, rendering them in remission and candidates to stop drug therapy. The researchers assessed patients’ MRD burden by RT-qPCR and ddPCR immediately after TKI discontinuation, then periodically for at least 3 years to detect relapse events and compare each method’s ability to measure MRD.

Based on the MRD testing results of the 60 patients who experienced molecular recurrence, patients who tested negative for MRD by RT-PCR and ultra-sensitive testing via ddPCR were the best candidates to stop TKI therapy.

• Of the patients with no BCR::ABL1 transcripts detectable by RT-qPCR or ddPCR, 10% relapsed.
• Of the patients whose BCR::ABL1 transcripts were undetectable by RT-qPCR and detectable by ddPCR, 64% relapsed.
• Of the patients with BCR::ABL1 transcripts detectable by RT-qPCR and ddPCR, 50% relapsed after TKI discontinuation.

These results highlight the importance of reflexive testing of borderline qPCR tests by ddPCR to assess any samples containing DNA or RNA below the limit of detection of RT-qPCR. By enabling clinicians to predict a patient’s risk of recurrence more precisely, this MRD testing strategy helps doctors reduce unnecessary treatments and improve the quality of life for some patients.

Case Study 2

MRD Testing Triggers Earlier Intervention

In another example, our lab works with patients with leukemia who have received a bone marrow transplant. We must monitor patients closely after the procedure for cancer recurrence. We perform whole genome sequencing to identify patient-specific DNA translocations that may serve as biomarkers for MRD testing, and we design primers for ddPCR assays around these translocations. Because the assays target a sequence that does not exist in nature, they need to be remarkably sensitive. This approach is currently being compared to other standard methods. We have also used ddPCR to monitor pathogenic variants found at diagnosis in patients with myelodysplastic neoplasms undergoing bone marrow transplantation to predict relapse. We find that ddPCR can detect the disease 4 months earlier compared to other methods.

Our work in this area is ongoing, but based on our findings, a highly sensitive assay design in combination with ddPCR provides a larger window of opportunity to alter a patient’s treatment plan as soon as an MRD-positive result is detected. In the future, we intend to study whether using MRD status to direct changes in treatments can benefit patients.

References

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2. Chin RI, Chen K, Usmani A, et al. Detection of Solid Tumor Molecular Residual Disease (MRD) Using Circulating Tumor DNA (ctDNA). Mol Diagn Ther. 2019;23(3):311-331. doi:10.1007/s40291-019-00390-5
3. Leukemia and Lymphoma Society. Minimal Residual Disease (MRD) Fact Sheet. lls.org/sites/default/files/National/USA/Pdf/Publications/FS35_MRD_Final_2019.pdf
4. Hao T, Li-Talley M, Buck A, Chen W. An emerging trend of rapid increase of leukemia but not all cancers in the aging population in the United States. Sci Rep. 2019;9(1):12070. Published 2019 Aug 19. doi:10.1038/s41598-019-48445-1
5. Vessies DCL, Greuter MJE, van Rootijen KL, et al. Performance of four platforms for KRAS mutation detection in plasma cell-free DNA: ddPCR, Idylla, COBAS z480 and BEAMing. Sci Rep. 2020;10(1):8122. doi.org/10.1038/s41598-020-64822-7
6. Larribère L, Martens UM. Advantages and Challenges of Using ctDNA NGS to Assess the Presence of Minimal Residual Disease (MRD) in Solid Tumors. Cancers (Basel). 2021;13(22):5698. doi:10.3390/cancers13225698
7. Apperley JF. Chronic myeloid leukaemia. Lancet. 2015;385(9976):1447-1459. doi:10.1016/S0140-6736(13)62120-0
8. Atallah E, Schiffer CA, Weinfurt KP, et al. Design and rationale for the life after stopping tyrosine kinase inhibitors (LAST) study, a prospective, single-group longitudinal study in patients with chronic myeloid leukemia. BMC Cancer. 2018;18(1):359. doi.org/10.1186/s12885-018-4273-1