Role of Liquid Biopsies in Monitoring Hematological Malignancies

Introduction:

Nowadays, assessing cancer mutational profiles is vital for patient management, stratification, and therapeutic decisions. Liquid biopsy is a developing approach for characterizing tumors through the isolation and analysis of various cancer-derived components that are released in any body fluids like urine, blood, liquor, or others. The role of liquid biopsies in monitoring minimal residual disease (MRD) in hematological malignancies is to look for the presence of alterations in nucleic acids, usually in peripheral blood or other biological fluids, which is being studied and optimized with increasingly innovative molecular techniques, such as next-generation sequencing (NGS) in the monitoring of MRD and other conditions.

What Is Liquid Biopsy (LB)?

Liquid biopsy (LB) tests the tumor's genomic profile using peripheral blood (PB) components. Various tumor constituents can be obtained from the blood, which can help develop individualized therapies, monitor the progression of the tumor, treat monitoring responses, and quantify minimal residual disease (MRD). This clinical tool has the advantage of being non-invasive. It can provide vital information about the burden of the disease, the tumor's heterogeneity, and evolution.

LB consists of various biological matrices, which include circulating tumor cells (CTCs), cell-free circulating nucleic acids (DNA- deoxyribonucleic acid), mRNA (messenger ribonucleic acid) micro-RNA (ribonucleic acid), or non-coding RNA, "tumor-educated platelets" (TEPs), and exosomes. In the early stages of tumorigenesis (a process in which normal cells convert into cancer cells or neoplastic cells), CTCs are usually shed into the bloodstream. CTCs thus represent an essential marker in early screening, diagnosis, and relapse detection. CTCs can be isolated from the blood using physical and biological properties such as tumor-specific surface antigens. Comparing genomic sequencing of individual CTCs with genomes of the primary tumor can provide new insight and help detect early metastasis (spread).

Several gene mutations like EGFR (epidermal growth factor receptor), KRAS (Kirsten rat sarcoma viral oncogene homolog), and HER 2 (human epidermal growth factor receptor 2) can also be evaluated from the CTCs, which can guide in the development of targeted therapy and thereby improve outcomes. The number of CTCs discovered is linked to the treatment efficacy and overall patient survival.

What Are Minimal Residual Diseases (MRD)?

MRD or minimal residual diseases are the minute amounts of remnants of the primary tumor present in the body after treatment, which also remain unidentified by high-resolution imaging techniques. The existence of MRD signifies possible short- or long-term relapse in cancer patients. It is an essential indicator of treatment response, so it becomes vital to check for the presence of MRD and treat it in the earliest identification stage, which will help prevent the development of untreatable metastatic illness.

How Does a Liquid Biopsy Differ From a Biopsy?

Unlike biopsies, liquid biopsies do not test tumor tissues directly. Instead, they test the evidence of a tumor. A biopsy is regarded as the ‘gold standard’ method and is considered the best procedure in the diagnosis of cancer. During a biopsy, a tissue sample is removed from a tumor, and the cells are tested in a laboratory to detect whether they are cancerous. On the contrary, a liquid biopsy identifies the signs of a tumor, like tumor DNA and tumor cells.

With a liquid biopsy, even if one has a tumor, there is no assurance that signs of that tumor will be identifiable in a single blood sample. However, liquid biopsies render valuable information about cancer cells that aid in treatment planning. Liquid biopsies can help detect pieces of tumor that break off and circulate in the bloodstream.

Liquid biopsies can identify or detect the following:

• Circulating Tumor Cells (CTCs): A CTC is a cancer cell from a tumor that travels in the bloodstream.

• Circulating Tumor DNA (CtDNA): It is a DNA fragment from a tumor cell that circulates in the blood. DNA contains the instructions or the genetic code, that controls the behavior of a cell.

What Is the Role of Liquid Biopsies in Monitoring Minimal Residual Disease (MRD) in Hematological Malignancies?

Liquid biopsy is a new test that has emerged recently with numerous potential groundbreaking uses in the treatment of cancer, with a few uses approved by the U.S. Food and Drug Administration (FDA). Research regarding its benefits is ongoing. The role of liquid biopsies in monitoring MRD are as follows:

• Minimal residual disease or measurable residual disease (MRD) refers to the persistence of malignant cells in patients who receive seemingly successful initial treatment. This MRD can potentially result in metastatic relapse at remote sites. MRD testing and monitoring are already widely used in hematological malignancies but still seem more challenging to apply in solid tumors due to the low blood levels of CTCs, which makes sampling challenging. MRD is considered an essential tool in myeloid malignancies and helps in prognostication (the process of estimating and communicating the expectations about a patient’s course of disease), disease monitoring, identifying early relapse, guiding post-remission treatment, and measuring the efficacy of the treatment.

• While different techniques have been developed for the assessment of MRD in myeloid malignancies, MRD test result indicates the presence of "MRD positive" or absence of "MRD negative" of detectable disease above certain thresholds, which may differ depending on the assay and the laboratory in which the testing is done.

• Flow cytometry (a laboratory technique in which lasers are used to study the physical and chemical properties of cells and particles in a fluid) evaluates the protein expression on the cell surface or in the cytoplasm using fluorescently labeled antibodies and can determine the immunophenotype (the process of detecting cells by the antigens or markers on their surface using antibodies) of cell lineages. Flow cytometry is best for MRD detection in acute myelocytic leukemia (AML- an aggressive and rare type of cancer that affects the blood and the bone marrow) and myelodysplastic syndromes (MDS- a group of cancers that target the bone marrow, causing it to generate fewer healthy blood cells). Quantitative reverse transcriptase polymerase chain reaction (RT-qPCR) includes converting the mRNA obtained from the specimen to cDNA through reverse transcription and then quantifying the cDNA via PCR. For instance, ABL1 is the most frequently expressed gene in chronic myeloid leukemia white cells.

• PCR (polymerase chain reaction) amplification followed by testing for overexpression of transcripts makes RT-qPCR one of the compassionate ways to detect MRD. With recent advancements in liquid biopsies, next-generation sequencing (NGS) monitors the response after myeloid malignancy therapy, especially AML. In NGS, DNA (deoxyribonucleic acid) fragments are amplified by PCR (from regions of interest), and sequences are compared with lines of reference. NGS helps detect mutations (changes) occurring in the exons like NPM1 (nucleophosmin 1) for AML.

What Are the Challenges and Future Directions Associated With MRD?

• Identification of MRD with liquid biopsy is challenging due to too-low concentrations of CTCs and ctDNA in the blood. This approach is also strongly limited by the availability of effective therapies in eradicating small amounts of residual tumors.

• To tackle the first difficulty, numerous molecular biology-based methods have been incorporated to obtain detailed genetic profiles of CTC and cDNAs. These methods can be classified into massively parallel sequencing technologies, next-generation sequencing (NGS), and digital genomic techniques such as digital PCR (dPCR), which provide biomarker and mutation information about specific tumors.

• The first known approved tumor-specific ctDNA-based MRD evaluating tool for solid tumors came to the market recently. Such new advancements in technology provide testing for a particular sequence of therapeutically relevant genes of the detected CTCs and ctDNA and customized blood tests to match the clonal mutations seen in each patient's tumor tissue.

• Studies reveal that MRD is an essential parameter of the depth of response, acting as a surrogate marker for more prolonged progression-free survival and overall survival. For potentially curative cancer diseases, liquid biopsy could have a vital role in early-stage patient management, preventing the development of incurable metastatic disease. Still, there are also directives to establish the role of these biomarkers in early-stage patients and establish clinical guidance for long-term post-remission therapies.

Conclusion:

Minimal residual disease (MRD) assessment through blood component sampling by liquid biopsies (LBs) is mainly being investigated in myeloid malignancies. Blood components are molecularly analyzed by flow cytometry or sequencing methods and can be enabled as robust equipment for prognostic and predictive purposes in myeloid malignancies. Evidence and more studies are evolving about quantifying and identifying cell-based and gene-based biomarkers in myeloid malignancies to assess treatment response. MRD-based acute myeloid leukemia protocol and clinical trials are now incorporating LB testing, and preliminary results are encouraging for its potential widespread use in clinics soon. MRD monitoring with the help of LB is not standard in myelodysplastic syndrome (MDS), but this is an area of active investigation. In the future, LBs can replace invasive methods like bone marrow biopsies.