Minimal Residual Disease (MRD) Detection

Minimal Residual Disease (MRD) Detection

1. Project Background

Cancer is one of the leading causes of death worldwide, and the recurrence and metastasis of solid tumors are key factors contributing to patient mortality [1]. In routine clinical practice, patients with solid tumors are typically monitored for disease recurrence through imaging studies (such as CT and MRI) following radical surgery or initial treatment. However, imaging technology has inherent limitations in detecting Minimal Residual Disease (MRD), as its sensitivity is insufficient to detect lesions when tumor burden is extremely low, often leading to delayed diagnosis and missed optimal intervention opportunities [2].

To standardize and promote the clinical application of MRD detection in China, the Chinese Society of Pathology and the National Center for Pathology Quality Control jointly published the "Consensus on Molecular Residual Disease Detection in Solid Tumors" (hereinafter referred to as the "Consensus") in 2024. This document explores the clinical application value, target populations, technical methods, strategies, timing selection, MRD detection standardization, and MRD detection reporting to guide MRD detection practice [6].

Core definition of the Consensus: Molecular residual disease (MRD), also known as minimal residual disease or measurable residual disease. In recent years, MRD detection technology for solid tumors has made significant progress. Multiple domestic and international clinical studies have demonstrated that MRD status is correlated with recurrence risk in patients with solid tumors, and can be used for prognostic evaluation and individualized treatment decision guidance. The Consensus clearly points out that ctDNA detection based on Next-Generation Sequencing (NGS) is currently the most commonly used method for solid tumor MRD detection (Category 2A recommendation), and has clear prognostic stratification clinical value (Category 2A recommendation) in colorectal cancer, non-small cell lung cancer, and breast cancer.

The Consensus clearly states that ctDNA detection based on Next-Generation Sequencing (NGS) is currently the most commonly used method for solid tumor MRD detection (Category 2A recommendation), and has clear prognostic stratification clinical value (Category 2A recommendation) in colorectal cancer, non-small cell lung cancer, and breast cancer.

In the context, the GeneRulor cfDNA MRD detection platform strictly adheres to the guiding principles of the "Consensus," employing advanced liquid-phase hybrid capture enrichment and NGS technology. It aims to achieve ultra-high-sensitivity detection of solid tumor MRD through ultra-deep sequencing and bioinformatics analysis of cfDNA, thereby providing earlier recurrence warnings, more accurate efficacy evaluations, and more individualized treatment guidance for clinical practice, ultimately improving patient survival prognosis.

2. Project Principles

The core technology of GeneRulor cfDNA™ is targeted high-throughput sequencing based on liquid-phase hybrid capture. Its fundamental principle involves using pre-designed biotinylated cytosine nucleotide probes targeting specific genomic regions to perform efficient and specific hybridization with cfDNA target regions in the sample. Subsequently, the target DNA fragments hybridized to the probes are captured and enriched from the complex background cfDNA using streptavidin magnetic beads, followed by library construction and high-throughput sequencing.

Liquid-phase hybrid capture technology can effectively enrich genomic regions of interest, significantly improving the depth and efficiency of sequencing, and is the key to achieving reliable detection of low-abundance ctDNA [4].

The entire detection workflow mainly includes the following key steps:

Figure 1. UMI-based Hybrid Capture for MRD Detection — Technical Workflow

(1)   cfDNA Extraction: Extracting high-quality cfDNA from peripheral blood plasma samples.

(2)   Library Construction:   Ligating sequencing adapters to both ends of the extracted cfDNA fragments and introducing unique Unique Molecular Identifiers (UMI). UMI technology assigns a "barcode" to each original DNA molecule, enabling precise traceability in subsequent data analysis, effectively removing PCR amplification bias and sequencing errors, and greatly improving the accuracy of low-frequency mutation detection.

(3)   Hybrid Capture:   Hybridizing the constructed library with the precisely designed probe library to specifically capture DNA fragments containing tumor-related genes or individual mutation sites.

(4)   High-throughput Sequencing: Performing ultra-high-depth sequencing (typically ≥30,000×) on the captured DNA library to ensure sufficient detection capability for extremely low-abundance ctDNA molecules.

(5)   Bioinformatics Analysis: Using a series of complex bioinformatics algorithms to perform deduplication, alignment, variant identification, and background noise filtering on the sequencing data, ultimately achieving precise interpretation of MRD status.

This pipeline supports two mainstream MRD analysis strategies to accommodate different clinical needs:

3. Detection and Analysis Advantages

Compared with traditional detection methods, GeneRulor cfDNA™ demonstrates unparalleled advantages in MRD monitoring.

Summary of Core Advantages:

(1)   Ultra-High Sensitivity:   Combined with UMI technology and ultra-deep sequencing (≥30,000×), precise capture of extremely low-abundance (<0.2%) ctDNA can be achieved, reaching detection levels as low as 10 ppm (parts per million) under certain conditions [2].

(2)   Non-invasive Safety:   Only a small amount of peripheral blood needs to be collected, avoiding the risks and pain associated with traditional tissue biopsy. High patient compliance enables high-frequency, continuous dynamic monitoring.

(3)   Dynamic Real-time:   The half-life of ctDNA in the blood is extremely short (approximately 1–2 hours), and its level changes can reflect increases in tumor burden in real time, providing a dynamic window for clinically evaluating therapeutic efficacy.

(4)   Comprehensive Representation:   Liquid biopsy can capture ctDNA from different metastatic sites, comprehensively reflecting the overall heterogeneity of the tumor, overcoming the sampling bias that may exist with single-site tissue biopsy.

(5)   Early Warning:   Extensive research has shown that ctDNA-MRD status conversion is on average several months to more than a year ahead of imaging detection of recurrence, gaining precious time for clinical re-intervention [5].

4. Application Scenarios

The GeneRulor cfDNA™ detection platform can be applied throughout the entire process of treatment and recovery for patients with solid tumors, providing key evidence for clinical decision-making.

(1)   Post-surgical Recurrence Risk Stratification:   After radical surgery, by detecting MRD status, patients can be precisely stratified into high-risk and low-risk groups. MRD-positive patients have extremely high recurrence risk, suggesting the need for more aggressive adjuvant therapy; while MRD-negative patients have good prognosis and may avoid over-treatment.

(2)   Adjuvant/Neoadjuvant Therapy Efficacy Evaluation:   During adjuvant or neoadjuvant therapy, by dynamically monitoring changes in ctDNA levels, therapeutic efficacy can be quickly and objectively assessed. Significant reduction or clearance of ctDNA levels usually indicates a good treatment response.

(3)   Immunotherapy Monitoring:   During immunotherapy with checkpoint inhibitors and other treatments, changes in ctDNA burden can serve as early biological markers for evaluating efficacy and predicting disease progression, assisting clinical decision-making.

(4)   Long-term Follow-up and Recurrence Monitoring:   For patients who have completed treatment and entered the follow-up period, regular MRD testing enables "sentinel" monitoring for disease recurrence; once ctDNA converts to positive, imaging confirmation and early intervention can be initiated.

5. Case Analysis

The GeneRulor cfDNA™ detection platform not only provides the final MRD-positive or MRD-negative result, but also provides throughout-process, transparent quality control indicators and analysis details to ensure the reliability and traceability of each report. The following demonstrates our analysis indicators, results, and their clinical guidance significance through key content of an example report.

A breast cancer patient required MRD dynamic monitoring after radical surgery and adjuvant chemotherapy to evaluate treatment efficacy and prognosis. We used the GeneRulor cfDNA™ pipeline to collect peripheral blood samples at multiple time points during the patient's treatment (baseline, Cycle 2, Cycle 3, Cycle 4) for ctDNA detection.

5.1 Dynamic VAF Changes: Visual Representation of Treatment Efficacy

By performing quantitative analysis on multiple tumor-specific mutations (such as KRAS G12D, TP53 R273H, APC Q1367*) carried in the patient's plasma cfDNA, we plotted the dynamic trend chart of VAF (Variant Allele Frequency).

Figure 2. MRD Dynamic Monitoring VAF Trend Chart. The chart clearly demonstrates the significant decline in VAF of multiple key mutations during the treatment process, visually reflecting the effectiveness of treatment.

Key Results Interpretation:

5.2 Clinical Guidance Significance: Precision Decision-Making Guided by VAF

As the core indicator of MRD monitoring, the dynamic changes in VAF provide key evidence for clinical decision-making:

(1) Efficacy Evaluation:   The dramatic decline in VAF from baseline to Cycle 2 (by more than an order of magnitude) indicates that the patient is highly sensitive to the current treatment regimen, and treatment is effective.

(2) Prognostic Assessment:   Achieving MRD-negative status at Cycle 4 (all mutation VAFs below the limit of detection, LOD) predicts that the patient has lower recurrence risk and longer disease-free survival (DFS).

(3) Treatment Adjustment:   If a plateau, increase in VAF, or new mutations are observed during monitoring, this may indicate disease progression or drug resistance, suggesting that clinicians need to consider adjusting the treatment strategy, such as switching chemotherapy regimens or combining targeted therapy.

(4) Long-term Follow-up:   For patients achieving MRD-negative status, follow-up intervals can be appropriately extended and unnecessary imaging examinations reduced, decreasing the patient's economic and psychological burden. For MRD-positive patients or those with rising VAF, monitoring frequency should be intensified.

Through precise VAF quantification and dynamic monitoring provided by the GeneRulor cfDNA™ pipeline, clinicians can achieve real-time, non-invasive assessment of tumor burden, thereby formulating more individualized treatment and follow-up plans to ultimately improve patient prognosis.

6. Service Content

7. Sample Requirements

To ensure the accuracy and reliability of detection results, please strictly comply with the following sample requirements:

(1) Sample Type:   Peripheral blood.

(2) Collection Tubes:   It is recommended to use dedicated cfDNA preservation tubes (such as Streck tubes) to prevent cell lysis from releasing genomic DNA and causing interference.

(3) Blood Volume:   8–10 mL of peripheral blood to ensure that sufficient plasma (≥4 mL) can be separated.

(4) Processing Time Limit:   Plasma separation should be performed within the specified time after collection (follow collection tube instructions).

(5) Storage and Transportation:   Plasma samples should be cryopreserved at −80°C and transported using dry ice.

(6) Tissue Samples (for Tumor-Informed strategy):   Fresh tissue surgically excised, fresh-frozen tissue, or FFPE tissue blocks/sections.

8. References

[1] Sung, H., Ferlay, J., Siegel, R. L., Laversanne, M., Soerjomataram, I., Jemal, A., & Bray, F. (2021). Global Cancer Statistics 2020: GLOBOCAN Estimates of Incidence and Mortality Worldwide for 36 Cancers in 185 Countries. CA: A Cancer Journal for Clinicians, 71(3), 209–249.

[2] Pantel, K., & Alix-Panabières, C. (2024). Minimal residual disease as a target for liquid biopsy in patients with solid tumours. Nature Reviews Clinical Oncology, 22, 65–77.

[3] NCCN Clinical Practice Guidelines in Oncology (NCCN Guidelines®). Colon Cancer. Version 2.2024.

[4] Aredo, J. V., Jamali, A., Zhu, J., Heater, N., et al. (2025). Liquid Biopsy Approaches for Cancer Characterization, Residual Disease Detection, and Therapy Monitoring. American Society of Clinical Oncology Educational Book, 45, e481114.

[5] Tie, J., Cohen, J. D., Wang, Y., Christie, M., et al. (2019). Circulating tumor DNA analysis guiding adjuvant chemotherapy in stage II colon cancer. New England Journal of Medicine, 380(23), 2261–2271.