How is molecular detection driving minimal residual disease testing?

Jeremiah McDole, Oncology Segment Manager of the Digital Biology Group at Bio-Rad Laboratories, discusses key trends and challenges on the topic of whether minimal residual disease testing can help more patients beat cancer.

According to the International Agency for Research on Cancer, almost 10 million individuals died from cancer worldwide in 2020, and approximately 19.3 million received new cancer diagnoses.¹ The annual global tally of new cases is expected to grow, fueled largely by aging populations and risk factors like smoking, poor diet, sedentary lifestyles and excessive body weight.¹ As such, experts predict a steady rise in incidence, leading to 28.4 million new cancer cases in 2040.¹ However, cancer assessment and treatment practices are evolving to improve outcomes for affected individuals. With this, there is a growing emphasis on measuring the small number of cancer cells remaining after curative treatment and post-adjuvant therapy —called minimal residual disease (MRD)—to predict risk of relapse and respond earlier in cases of recurrence.

Gold standard approaches, primarily imaging techniques, used to help diagnose and monitor cancer, cannot accurately measure MRD; instead, molecular tests are used to detect tumour cell biomarkers. As ultra-sensitive detection methods like droplet digital PCR (ddPCR) have entered the market, mounting data demonstrates that highly accurate MRD testing can provide vital insight into patients’ treatment trajectories, accelerating its use in the clinic.

The growing clinical role of MRD detection

Clinicians can use liquid biopsy to noninvasively measure MRD within patients. Tested samples are generally taken from blood, where tumour-specific biomarkers, usually detectable nucleic acid mutations within circulating tumour DNA (ctDNA), reside. MRD testing has been used for years to assess hematological malignancies, which by nature circulate in the blood. More recently there has been a push to use MRD testing to evaluate the minute MRD signals of solid tumours in the blood, too—a field of MRD testing made possible by the development of ultra-sensitive detection methods. As clinical trials report new successes, there has been growing adoption of new testing practices by clinicians. Fueled by this growing body of evidence, the clinical role of MRD testing continues to expand.

When a patient is suspected of having cancer, they must first undergo diagnostic procedures to image and molecularly characterise their tumour. Then, they may receive neoadjuvant therapy to reduce tumour size prior to receiving a curative treatment such as tumour resection, radiotherapy and/or chemotherapy. Molecular tests at these early stages may inform clinicians of tumour load and early prognosis. Subsequently, MRD measurements are taken after curative treatments to better understand a patient’s prognosis and stratify risk. In some cases, this testing can indicate the need for post-adjuvant therapy or, in the case of blood cancers, long-term monitoring.^{2, 3} Overall, MRD testing can improve outcomes by enabling doctors to deliver more personalised care than is possible through less sensitive techniques. For some high-risk patients, it indicates a clear need for adjuvant therapy to prevent recurrence, but for others who have responded well to therapy, it can allow doctors to reduce or avoid unnecessary treatment.

Before the advent of liquid biopsy, patients were given a prognosis and monitored for relapse primarily via imaging surveillance and tissue biopsy. This approach has several drawbacks. For instance, only visible tumours can be detected, MRD information cannot be captured, and the procedures are time-consuming, invasive and expensive. Now, liquid biopsy offers a superior method for MRD detection. Because liquid biopsy can capture MRD information that would have been invisible via imaging, it can provide critical information about a patient’s disease state much earlier. It also reduces the number of costly, invasive procedures a patient must undergo.

The role of MRD detection in the clinic is expanding as biomarker research advances. Some types of cancer are associated with common genetic mutations (i.e., KRAS for colorectal, lung and pancreatic cancer,⁴ EGFR in metastatic lung cancer,⁵ BRAF and NRAS for metastatic melanoma,⁶ and BCR-ABL1 for chronic myeloid leukemia⁷). But for other types, each patient’s disease has a unique mutational profile. As a result, the MRD testing approach varies by cancer type. Inexpensive commercial kits may be available to test MRD in cancers with well-established biomarkers, but the process is more expensive and complex in cancers without these clear molecular targets. Oftentimes, laboratories must evaluate the patient’s tumour by next generation sequencing (NGS) to establish the mutational profile. Biomarkers are then selected from the NGS data to become the targets of personalised polymerase chain reaction (PCR)-based tests.

A rising demand for MRD testing

As the number of people with cancer increases globally, the medical community is turning to MRD testing, now equipped with better tools and more biomarkers to guide clinical decision making.⁸ The rising annual incidence of hematological malignancies (such as leukemia, lymphoma and myeloma) is outpacing that of solid tumours, as exemplified by the doubling of new leukemia cases in the United States in the last two decades.⁹ Thus, the area within oncology in which ctDNA testing was first discovered will continue to be a major driver of the MRD market moving forward.¹⁰ On a positive note, treatments are increasingly successful for some cancer types, leading to a larger number of surviving patients who must be monitored long-term for relapse via MRD testing.⁹

Improved education and awareness among patients and caregivers about MRD testing has also contributed to the growth of the MRD testing market.¹¹ Much of this centers around how sensitive tests and careful monitoring can result in significantly better outcomes, leading to better compliance. Simultaneously, heads of testing labs and clinicians are gaining a deeper understanding of the MRD testing technologies available and can select based on sensitivity, accuracy, cost and speed. This awareness is leading to rising demand for affordable technologies that can quickly deliver exceptionally accurate and reproducible results with minimal analysis complexity.

Molecular tests to detect and quantify MRD

While MRD testing platforms have advanced in recent years, older technologies are still common-place. Among these are PCR-based DNA and RNA testing methods, such as quantitative PCR (qPCR) and quantitative reverse transcriptase PCR (RT-PCR). qPCR and RT-qPCR methods were developed many years ago and are commonly used in the clinic because equipment is readily available and the techniques are familiar. However, these methods require preparing standard curves to interpret results. This requirement introduces the potential for human error, resulting in a detection limit of about 0.1-1.0 percent of a target gene species.² ddPCR reactions do not require standard curves. A sample is partitioned into approximately 20,000 droplets, with one or a few DNA fragments in each. In a thermocycler, amplification occurs only in droplets that contain the target DNA. When the reaction is complete, a droplet reader counts the positive droplets based on a fluorescent signal they emit. That number is then used to calculate the precise number of target DNA molecules in the original single sample, with a limit of detection down to 0.01%.^{2, 12} Ultimately, this provides a simplified workflow and produces ultra-sensitive, accurate results that do not require advanced bioinformatics.

Beyond PCR-based methods, many labs leverage NGS as part of their MRD workflow. Targeted and untargeted next-generation sequencing (NGS) techniques allow for the assessment of many biomarkers at once.¹³ NGS technology can sequence millions of DNA fragments in a massively paralleled reaction and align them to a reference sequence to uncover previously unknown mutations. When paired with ultra-sensitive methods such as ddPCR, the combined data generated offers greater research and clinical value than either standalone system.

ddPCR^{14, 15}

• Select target number
• Lower cost per run
• Sample extraction to answer in ~8 hours
• Highest sensitivity
• Simple workflow: Small technician burden
• Small data volume: Simple interpretation

NGS^{14, 15}

• Assess large volume of targets
• High cost per run
• Sample extraction to answer in ~24 hours (~36+ hours for hybridization capture protocols)
• High sensitivity (depending on platform and assay)
• Complex workflow: High technician burden
• Large datasets: Advanced bioinformatics needed to interpret results; Storage of large datasets a growing concern

While assessment by NGS is highly valuable and often necessary for MRD testing, ddPCR offers several practical advantages when processing high volumes of samples for which biomarkers have been established (summarised above).

Ultra-sensitive MRD testing in action

Many studies have demonstrated the benefits of enhanced sensitivity for MRD testing. The publications below provide a few examples.

For example, treating chronic myeloid leukemia (CML) with tyrosine kinase inhibitors (TKIs) has proven to be a highly effective therapy, expanding life expectancy in patients from about 6 years to a full lifespan.¹⁶ However, side effects from TKI treatment can reduce quality of life. To address this issue, the LAST Study, the most extensive U.S.-based study of its kind, used MRD monitoring of the CML biomarker BCR-ABL1 to establish how to identify patients who could safely stop TKI therapy because they were at low risk of relapse.⁷ The prospective study examined 172 patients who had been receiving TKI therapy for three or more years. They had achieved major molecular response to the TKIs (defined as BCR-ABL1 transcript levels below 0.01 percent), and their BCR-ABL1 transcript levels had stayed at or below this level for two or more years, indicating that they were in remission and therefore candidates to stop drug therapy. Patients’ MRD burden was assessed by ddPCR and RT-qPCR immediately after TKI discontinuation and monitored over at least three years to detect relapse events.

Based on data from the 60 patients who experienced molecular recurrence, ultra-sensitive testing via ddPCR is key for identifying the strongest candidates to stop TKI therapy. 50 percent of patients with BCR-ABL1 transcripts detectable by RT-qPCR after TKI discontinuation relapsed. 64 percent of those with BCR-ABL1 transcripts undetectable by RT-qPCR but detectable by ddPCR relapsed. In contrast, only 10 percent of patients with no BCR-ABL1 transcripts detectable by RT-qPCR or ddPCR relapsed. The results of the LAST study highlight the importance of reflexive testing of borderline qPCR tests by ddPCR to assess for low levels of biomarkers below the limit of detection of RT-qPCR. This MRD testing strategy can predict risk of recurrence more precisely, making it possible to reduce unnecessary therapy to improve quality of life for some patients.

Similarly, MRD measurement of BCL1/IGH rearrangements can indicate prognosis for mantle cell leukemia (MCL) patients, but over half of positive MRD samples fall below the quantitative range of qPCR (<0.01 percent target mutation) and cannot inform clinical decision making. Therefore, one study put the sensitivity of qPCR, ddPCR and flow cytometry to the test, evaluating 416 samples where a majority fell below qPCR’s quantitative range.¹⁷ While all three techniques yielded similar results for samples containing <0.01 percent mutation, differences became apparent at lower mutational frequencies. Flow cytometry was considered the least expensive but also least sensitive technique. In contrast, ddPCR was found to be most sensitive, and particularly valuable for measuring samples carrying 0.01–0.001 percent target mutation.¹⁷ Overall, ddPCR’s higher sensitivity meant that only 12 percent of samples were unquantifiable, making it possible to accurately evaluate MRD for more patients than would have been possible with qPCR alone. As a result, the study proposed guidelines on using ddPCR in MRD testing to evaluate MCL patients, enabling doctors to make better-informed decisions based on MRD.

MRD ambiguity can also prove problematic for solid tumour analysis such as in patients with colon cancer. Surgical resection can be curative, but there is a high risk for relapse.¹⁸ Pathological staging of tumour tissue is often used to predict risk, but it is imprecise, often leading to under- and over-treatment. Therefore, a prospective study of 150 patients sought to determine if MRD testing could better detect risk of relapse.¹⁸ The researchers used NGS to characterise mutations within each patient’s tumour, selecting at least two biomarkers for MRD testing via ddPCR for optimal accuracy. After surgery, they found that the presence of MRD was a good predictor of early relapse. In the future, ctDNA testing of patients with localised colon cancer may help clinicians better tailor adjuvant strategies to their patients’ needs: reducing treatment for those likely to be cured and starting treatment earlier for those at higher risk.

Future Applications and Growth of MRD Testing

These and other studies demonstrate a trend of using ultra-sensitive MRD testing to make cancer treatment more precise, effective and streamlined. Each new study helps build on the already established groundwork for utilizing MRD testing more routinely and across a growing number of cancer types.

Although MRD testing can provide incredible insight, several barriers have slowed full clinical adoption. MRD tests typically must assess one or a few biomarkers. For some cancers with well-defined biomarkers, this is relatively easy and cheap. For others, it is costly because NGS must be utilised establish personalised biomarkers. This challenge is especially relevant when considering MRD testing in solid tumour cancers. Furthermore, the approach to selecting biomarkers and measuring MRD is not standardised: different labs employ various NGS platforms, qPCR, RT-PCR, ddPCR and a host of other tests, leaving questions on how to standardise result interpretation and readout. However, many thought leaders recognise these barriers and are systematically working to resolve these challenges as the need for MRD testing grows.

Ultra-sensitive methods for MRD detection have proven advantageous in numerous clinical studies. Efforts continue toward standardization and will help expand what can be gleaned, leading to more widespread adoption of the available techniques. Simultaneously, as biomarker research uncovers more mutations common to whole cancer types, these may be converted into inexpensive, commercial tests.

As MRD testing strategies become better established and researchers uncover new uses, expanding applications in the clinic will match pace. However, clinical studies must continue to define standardised diagnostic metrics such as MRD presence or quantity to understand how these translate into prognosis and risk. As medical advances continue to drive growth, the momentum will promote increased awareness and education amongst the clinical community and patients about MRD testing, further unifying the field and leading to better patient outcomes.

About the author

McDole received his Ph.D. in neuroimmunology from the University of Cincinnati and spent his post-doctoral years on a number of successful research projects in the immunology depart at Washington University School of Medicine in St. Louis.

References

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