Cancer is the second leading cause of death in the United States, with an estimated > 1.8 million new diagnoses in 2020. Various topics within the field of cancer screening, testing, and management have been described previously in this series.  The field of cancer diagnostics and treatment is evolving at a rapid pace, even amid the current global COVID-19 pandemic. Recently two topics within oncology have been the subject of research and clinical application – minimum residual disease and tumor mutation burden. Malignant tumors shed cells and biomarkers including circulating tumor DNA (ctDNA) that can potentially be measured from a blood specimen. The purpose of this article is to explore how ctDNA technology is used to assess minimum residual disease and tumor mutation burden when treating a patient for cancer.

Minimum residual disease (MRD) is a function of cancer therapy efficacy. MRD is a measurement of residual cancer cells after the patient has been treated. These cancerous cells may be undetected by traditional testing methodologies. MRD is a predictor of relapse. Even if only one cancer cell survives treatment, it has the potential to cause the patient to relapse, so the optimization of testing for MRD with the highest sensitivity and lowest detection rates is crucial.

Historically, flow cytometry has been the methodology for measuring MRD, which utilizes fluorescently labeled antibodies to differentiate and sort cells in a specimen of biological fluid. Cancerous cells may be detected in a mixed-cell specimen with this technique. Analytically validating ctDNA methodologies involves considerable time, effort, and resources from the commercial lab that wants to add a clinical ctDNA test to their testing menu.  Some studies have reported that further research is needed, however, to validate the clinical utility of ctDNA testing.

A prospective multicenter cohort study demonstrated actionable information when determining MRD with ctDNA testing techniques. One hundred and twenty-five patients enrolled with colorectal cancer (stages I, II, or III) had ctDNA testing before and after surgery. Patients who were ctDNA positive 30 days post-operatively were at a seven-fold increased risk for relapse. Similarly, patients who were positive for MRD by ctDNA testing after adjuvant chemotherapy were at a seventeen-fold risk for relapse. Guardant Health commercialized their Guardant Reveal test in the first quarter of 2021 for detecting MRD via ctDNA. It has a 91% sensitivity by testing ctDNA for genetic changes and methylation patterns. Results are available in seven days, and there is no need for a tissue biopsy. As research progresses, it is likely other companies will offer similar ctDNA testing products for MRD.

Tumor Mutation Burden (TMB) is defined as the number of somatic mutations within a malignant tumor and is measured as the total number of mutations per coding area. TMB is the result of internal factors such as changes in DNA replication or repair or genes and external influences including smoking or UV light exposure, among others. For a gene to be included in TMB, the genetic mutation must result in an altered protein.  Unfortunately, cancer showing high TMB may be associated with poor outcomes. These modified proteins due to genetic mutations can be targeted for immune therapy. Pharmaceutical companies are using TMB in their clinical trials to validate therapies in different types of cancer tumors.

ctDNA is being researched to quantify TMB. Historically, genetic testing for TMB uses a biopsy of the tumor. Biopsy technology has been available for decades and there are established protocols for testing biopsies. Nevertheless, biopsies are invasive, and any test results may not capture the genetic heterogeneity of the entire tumor. Also, radiological assessment as may be needed for biopsy localization increases patient radiation burden. ctDNA methods are non-invasive, may be a better genetic representation of the entire tumor, and can be performed serially to measure TMB progression. On the other hand, ctDNA may not be informative for patients with a low tumor burden and could be below assay detection limit. A study comparing traditional biopsy with ctDNA testing in patients with non-small cell lung cancer found ctDNA methods to be feasible, precise, and reliable. The study also reported ctDNA was able to obtain results for 81% of participants vs. 63% for the biopsy technique.

Currently, there are multiple ongoing clinical trials sponsored by different commercial labs investigating ctDNA technology for cancer screening. GRAIL, one of these ctDNA companies, reported preliminary results from a clinical trial that are very encouraging. A study with over 6000 patients enrolled demonstrated ctDNA technology was able to detect 12 different cancer types in very early stages. These early cancers may be missed by traditional screening methodologies. Additionally, GRAIL has recently launched their Galleri test, which is designed to be a “precancer” screen. Providence health has partnered with GRAIL to offer the Galleri test to patients with the goal of early detection, intervention, and improved patient outcomes.

There is a tremendous amount of research being performed on ctDNA testing technology for cancer screening, assessment, and management. MRD and TMB are metrics used by clinicians that may impact management, surgery, or therapeutic options for the patient. As ctDNA technologies improve and new tests are validated, there will likely be an increase in choice and availability to healthcare professional for different ctDNA tests by multiple labs. It is likely some of these new tests may be specific to different types of cancer, while others are more of a broad-spectrum screen for multiple caner types. Additionally, the goal of early intervention may be facilitated by “precancer” tests which are designed to detect cancerous ctDNA that may not be detected by other types of screening. All of these ctDNA testing methodologies have the same objective – to improve outcomes for patients diagnosed with cancer.



Kostopoulos IV, Ntanasis-Stathopoulos I, Gavriatopoulou M, et al. Minimal Residual Disease in Multiple Myeloma: Current Landscape and Future Applications With Immunotherapeutic Approaches. Front Oncol. 2020 May 27;10:860.

Geeurickx E, Hendrix A. Targets, pitfalls and reference materials for liquid biopsy tests in cancer diagnostics. Mol Aspects Med. 2020 Apr;72:100828.

Jung D, Jain P, Yao Y, Wang M. Advances in the assessment of minimal residual disease in mantle cell lymphoma. J Hematol Oncol. 2020 Sep 24;13(1):127.

Fenizia F, Pasquale R, Roma C, et al. Measuring tumor mutation burden in non-small cell lung cancer: tissue versus liquid biopsy. Transl Lung Cancer Res. 2018 Dec;7(6):668-677.

Boonstra PA, Wind TT, van Kruchten M, et al. Clinical utility of circulating tumor DNA as a response and follow-up marker in cancer therapy. Cancer Metastasis Rev. 2020 Sep;39(3):999-1013.

Si H, Kuziora M, Quinn KJ, et al. A Blood-based Assay for Assessment of Tumor Mutational Burden in First-line Metastatic NSCLC Treatment: Results from the MYSTIC Study. Clin Cancer Res. 2021 Mar 15;27(6):1631-1640.

Kujala J, Hartikainen JM, Tengström M, et al High mutation burden of circulating cell-free DNA in early-stage breast cancer patients is associated with a poor relapse-free survival. Cancer Med. 2020 Aug;9(16):5922-5931.

Beer TM. Novel blood-based early cancer detection: diagnostics in development. Am J Manag Care. 2020 Nov;26(14 Suppl):S292-S299.

Reinert T, Henriksen TV, Christensen E, et al. Analysis of Plasma Cell-Free DNA by Ultradeep Sequencing in Patients With Stages I to III Colorectal Cancer. JAMA Oncol. 2019 May 9;5(8):1124–31.

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