Declaring a patient "cancer-free" is complex, carrying hope tinged with uncertainty. Why do some patients enjoy long-term remission while others face the challenge of relapse? Current studies increasingly link this variance to minimal residual disease (MRD). 

MRD, also termed molecular or measurable residual disease, represents a small number of cancer-specific molecules that persist in the body post-cancer treatment. These molecules, such as proteins or DNA, are undetectable through standard imaging methods like PET or CT scans and may exist without causing any apparent clinical symptoms.

ctDNA analysis

ctDNA is shed by dying tumor cells into the bloodstream and has a short half-life of 35 minutes, reflecting real-time cancer dynamics. Assessment within 3 days post-R0 resection is therefore recommended.

Liquid biopsies detect ctDNA in biological fluids, analyzing the variant allele fraction (VAF) to quantify cancer cell DNA presence, as reported by Chinese researchers in Frontiers in Genetics.

Zhu L, Xu R, Yang L, et al. Minimal residual disease (MRD) detection in solid tumors using circulating tumor DNA: a systematic review. Front Genet. 2023;14:1172108.

Diagnostic approach

MRD testing employs tumor-informed and tumor-agnostic methods, as discussed in a 2023 review from Oncology Reports.

Chen H, Zhou Q. Detecting liquid remnants of solid tumors treated with curative intent: Circulating tumor DNA as a biomarker of minimal residual disease (Review). Oncol Rep. 2023;49(5):106.

Detection method

The Oncology Reports authors state that VAF may be as low as 0.01%–0.10% in post-surgical or early stage cancers. Thus, sensitive and specific methods are needed to distinguish trace cancer-derived DNA from non-cancerous mutations in blood. PCR and next-generation sequencing (NGS) are employed to address this issue. Examples are cited in Frontiers in Genetics:

• Droplet Digital PCR (ddPCR) divides DNA samples into droplets for individual analysis after PCR, achieving high sensitivity at a lower cost but limited to detecting about 0.1% of gene targets.

• PCR amplicon-based NGS, capable of examining multiple DNA sequences simultaneously, uses unique molecular identifiers (UMIs) for increased accuracy. However, it is restricted to specific genomic regions and can exhibit allele amplification bias.

• Hybridization capture-based NGS uses biotinylated probes for DNA hybridization, excelling in concurrent mutation detection with sensitivity up to one part-per-million.

• Whole genome sequencing (WGS) and whole exome sequencing (WES) apply NGS to ctDNA, with WES being more cost-effective but limited to exonic regions.

Role in solid tumors

Following the promising results of MRD testing in hematological malignancies, recent studies have also established its utility across multiple solid tumors. MRD analysis carries high sensitivity (88%–100%) and specificity (98%–100%) in detecting lung, breast, esophagus, and bladder cancer relapse, with significant lead times over traditional radiographic imaging.

CMS. MolDX: Minimal Residual Disease Testing for Cancer. L38835. Medical Coverage Database. October 17, 2023.

The Frontiers in Genetics review highlights some noteworthy milestones of MRD testing:

Lung cancer

In post-surgery non-small-cell lung cancer (NSCLC) patients, MRD predicts recurrence risk, disease progression, prognosis, and survival rates. The 2023 meta-analysis reported in BMC Medicine, involving 1,251 patients, found that ctDNA was a reliable detector for relapse in NSCLC with high specificity (86%–95%) and moderate sensitivity (41%–76%).

Breast cancer

For early stage, resectable breast cancer, MRD can monitor tumor load, guide therapy, and predict recurrence risk, especially in high-risk triple-negative cases. In advanced stages, ctDNA complements radiological assessments.

Colorectal cancer

MRD helps select appropriate candidates with stage II and III colorectal cancer who might benefit from additional therapy. The GALAXY study, for instance, indicated that ctDNA levels declined more rapidly in patients receiving adjuvant chemotherapy, suggesting its utility in treatment monitoring.

Other solid tumors

• MRD predicts outcomes in locally advanced, unresectable, or metastatic gastric cancers post-chemotherapy. 

• Combining ctDNA and alpha-fetoprotein detection improves recurrence risk prediction post-surgery for early and intermediate-stage liver cancer patients.

• In pancreatic cancer, ctDNA detects disease progression earlier than radiological imaging.

• MRD helps determine the necessity of perioperative chemotherapy in upper tract urothelial carcinoma.

Challenges and future prospects

Researchers from Washington University School of Medicine expressed their view of the current state of ctDNA assessment: "The most fundamental challenge in the analysis of ctDNA is its scarcity," they wrote.

Semenkovich NP, Szymanski JJ, Earland N, et al. Genomic approaches to cancer and minimal residual disease detection using circulating tumor DNA. J Immunother Cancer. 2023;11(6):e006284.

The Frontiers in Genetics authors identified other problems, such as the fact that tumor-derived ctDNA load can vary across individuals and different cancer types. Factors like low DNA shedding and metastasis location cause false-negatives, while false-positives can result from DNA fragments of clonal hematopoiesis of indeterminate potential (CHIP) or benign hematopoietic cells.

Additionally, inconsistencies in ctDNA results can be due to variable sample handling and lack of standardized testing, complicating the interpretation of results.

There is a growing interest in the role of epigenetic signals, like DNA methylation alterations and fragmentation patterns, to differentiate ctDNA from benign circulating cell-free DNA, with the potential for reducing false-positives.

Johnston AD, Ross JP, Ma C, et al. Epigenetic liquid biopsies for minimal residual disease, what's around the corner? Front Oncol. 2023;13:1103797.

Despite its challenges, post-resection MRD analysis can guide adjuvant chemotherapy regimens and reduce the risk of relapse.