Minimal residual disease (MRD) and Liquid biopsy

Minimal or microscopic residual disease (MRD) is defined as a very small number of carcinoma cells which remain in the body during or after treatment of cancer. The use of MRD in treatment selections got FDA approval in 2018 in the context of B-cell precursor acute lymphoblastic leukemia. Tumor burden decreases with response to treatment and cancerous cells become undetectable in imaging or clinical testing. However, the cancerous cells remain in the body after treatment and play a role in recurrence or relapse of disease. The capability to detect MRD in non-hematological cancers may certainly be advantageous for both patients as well as clinicians using liquid biopsies. The main goal of MRD detection is to determine escalation or de-escalation of treatment for cancer patients. MRD positive patients may have the potential to improve disease-free and overall survival by efficient treatment of MRD via intensifying the treatment. On the other hand, if patients are MRD negative, then omission of adjuvant therapy could reduce treatment related adverse effects, improve quality-of-life and reduce the financial burden of patients without affecting survival rates in cancer.

New clinical trials are under the process of gathering information on diverse tumor types in order to establish the importance of MRD detection for the selection of personalized medicine after curative local therapy. The detection of MRD is limited by reliable detection methods as it cannot be assessed by standard imagery or clinical examination. Liquid biopsies, which are highly sensitive methods, may provide reliable detection of MRD.

Liquid biopsy is defined as a diagnostic tool for the diagnosis of cancer using circulating analytes derived from tumors that are circulating in the bodily fluids (blood, saliva, cerebrospinal fluid and urine). The detection of MRD could be performed by using cfDNA as a biomarker in liquid biopsy. The cfDNA is released into the bloodstream by both active and passive secretions such as due to cellular degradation, apoptosis and necrosis. The US-FDA approved the first cfDNA blood liquid biopsy of epidermal growth factor receptor (EGFR) genotyping assay for lung cancer patients in 2016. It was shown that the size selection of cfDNA fragments can be used to increase the amount of ctDNA (a small fragment of cfDNA) for diagnosis of cancer. Digital PCR, q PCR, targeted DNA sequencing, whole genome sequencing and NGS are the primary molecular based approaches for the isolation of cfDNA. The ability to monitor tumor burden and MRD are correlated with the rate of shedding of cfDNA, which differs between cancer type, stage and clinical situation.

1. Analytes for detection of MRD with liquid biopsy

1.1. Cell free DNA (cfDNA)

The concentration of cfDNA in cancer patients can be anywhere from 50 times more than the normal range. Clinical data has shown that cfDNA plays an important role in MRD monitoring and prevents relapse of multiple myloma. Another study has shown that the cfDNA-based methods for MRD detection are less expensive and a quicker option in relapse cases of chronic lymphocytic leukemia patient population. The cfDNA quantification with qPCR demonstrates that length of cfDNA fragments is associated with the response of patients to MRD treatment. Advancement in the detection of cell free DNAs based on a two-step sequencing method. The first step is ultra-low pass whole genome sequencing (UPLS), which is cost-effective and requires a small amount of samples as compared with NGS for the identification of tumor DNA fraction from blood plasma (5-10%).

1.2. Mitochondrial circulating tumor DNA (mtDNA)

Mitochondrial circulating tumor DNA (mtDNA) is also released by tumor cells and could potentially be a biomarker for diagnosis and management of cancer. The concentration of mtDNA may be raised in some cancers, while not in all types of cancer. Until now, MRD detection through mtDNA mutations has provided promising results in leukemia patients. Additionally, more clinical trials have to be performed for establishment of the role of mtDNA in detection of MRD cases of cancer and improve survival rates.

1.3. Methylation pattern

The mis-methylation process which is observed in cancer cells is responsible for tumorigenesis and it occurs earlier during tumorigenesis. In normal cells, methylation occurs in a specific pattern and is controlled by regulatory elements. However, in cancer cells, methylation patterns are disrupted, which leads to a down-regulation of the expression of tumor suppressor genes and an upregulation of expression of oncogenes.

Clinical studies have also shown that the absence of met-cfDNA or met-ctDNA decreases during treatment. However, they were found (in very low concentration in plasma) in the bloodstream after treatment correlating them with MRD and with a higher risk of relapse of cancer. Methylation-based plasma biomarkers of cfDNA and ctDNA could be powerful biomarkers for the detection of MRD in cancer patients as they are detected in plasma at an early stage. In one clinical study, MYO1G, which is a methylated marker of cfDNA, was found significantly in a plasma sample of colorectal carcinoma patients. So MYO1G can be used for the detection of MRD cases of colorectal cancer.

All these biomarkers require established sensitive detection methods to implement it in clinical practice. Various clinical trials are currently ongoing to evaluate methylation patterns in the bloodstream as a cancer detection tool (NCT04814407, NCT04511559) along with monitoring disease and detecting the presence of MRD (NCT03634826, NCT03737539).

1.4. Non-coding RNA

RNA is a single strand ribonucleic acid and can be divided into coding RNAs and non-coding RNA (ncRNA). Coding RNA is also called messenger RNA (mRNA). ncRNA is further subdivided into housekeeping RNA and regulatory ncRNA. Housekeeping RNA has again subdivision includes transfer-RNA and ribosomal-RNA. Regulatory ncRNA is also subdivided on the basis of their structures like long-ncRNA (>200 nucleotides also called IncRNA), circular-RNA and functional ncRNA. Functional ncRNA includes micro-RNA (miRNA), small-ncRNA, small nuclear-ncRNA and Piwi-interacting-RNA (piRNA). All these types of RNAs are found in the nucleus and cytoplasm of cells and released into the bloodstream or in other body fluids through tumor, which can be detected as biomarkers with liquid biopsy for diagnosis of cancer. Among all, the most investigated ncRNAs are miRNA and IncRNA. The miRNAs can act as a MRD biomarker because they modulate the expression of genes by binding to mRNA and silencing translation by which cancer cells express specific miRNA signatures that can be detected in the bloodstream with liquid biopsy to detect MRD. Multiple studies are currently under the process to assess the potential role of miRNA in several cancers like breast cancer (NCT04720508), pancreatic cancer (NCT04406831), prostate cancer (NCT04835454), head & neck cancer (NCT04305366), and many others. IncRNA has also been investigated as a potential biomarker of tumor cells in many cancers and can be detected in the bloodstream. It is correlated with tumor stage and treatment response. One clinical trial in breast cancer was based on an assessment of a risk of relapse by utilizing an lncRNA biomarker (NCT02641847) and hence, IncRNA could also be evolved as a predictive biomarker for the detection of MRD. Further, more clinical studies are required to establish a proper liquid biopsy method for the detection of IncRNA as a biomarker in MRD.

1.5. Exosomes

Exosomes are extracellular vesicles (EVs) that have a phospholipid bilayer membrane containing analytes (DNA, RNA, cytosolic & transmembrane proteins and cytosolic metabolites) derived from tumor cells and circulating in the bloodstream. Exosomes are key players in tumorigenesis by allowing inter-cellular communication which induces epithelial-to-mesenchymal transition (EMT), invasion and migration of cancerous cells, and a tolerant immune environment. Studies have shown that cancer cells may produce up to 20-fold more exosomes than non-cancerous cells. Cancer-specific DNA and RNA modifications are also found in exosomes. Therefore, MRD can also be detected through cancer exosomes, which can be isolated using cancer-specific proteins expressed on their surface from plasma using liquid biopsy. However, standardized isolation and quantification methods are still lacking. Many clinical trials are ongoing to evaluate the diagnosis of tumors and the detection of MRD through exosomes.

The detection of MRD through analytes of blood is imperative by using liquid biopsy, which could help in personalized medicine selection of MRD patients. However, several technical problems need to be addressed before the implementation of these in clinical practice. More clinical studies are required for evaluation of whether treatment escalation improves cancer outcome in MRD-positive patients and treatment de-escalation is safe in MRD-negative patients. These future trials will be helpful in the determination of the role of liquid biopsy in clinical practices for detection of MRD.

References

1. Honore N et al. 2021. Liquid Biopsy to Detect Minimal Residual Disease: Methodology and Impact. Cancers; 13(21): 5364.

2. De Rubis G et al. 2019. Liquid Biopsies in Cancer Diagnosis, Monitoring, and Prognosis.  Trends Pharmacol Sci. 40(3):172-186.

3. clinicaltrials. gov access on May 2021.

4. Larribere L and Martens UM. 2021. Advantages and Challenges of Using ctDNA NGS to Assess the Presence of Minimal Residual Disease (MRD) in Solid Tumors. Cancers (Basel); 13(22): 5698.

5. Thakral D et al. 2020. Cell-free DNA for genomic profiling and minimal residual disease monitoring in Myeloma- are we there yet? Am J Blood Res. 10(3): 26-45.

6. Gina Mauro. 2021. cfDNA-Based MRD Assay Is Feasible for Use in Relapsed/Refractory CLL. Targeted Therapies in Oncology. 1(1).

7. Furstenau M et al. 2021. Cell-free DNA (cfDNA)-based serial minimal residual disease assessment in patients with chronic lymphocytic leukemia treated with time-limited obinutuzumab, acal-abrutinib and venetoclax. Presented at: 2021 International Workshop on CLL; September 17-20, 2021; virtual. Abstract 1084129.