CIRCULATING TUMOUR DNA (cTDNA)- The promising tumour biomarker in liquid biopsy

Circulating tumour DNA (ctDNA) is single- or double-stranded DNA released by the tumour cells into the blood and it thus harbours the mutations of the original tumour.

In recent years, liquid biopsy based on ctDNA analysis has shed a new light on the molecular diagnosis and monitoring of cancer.

Tissue biopsy is the standard diagnostic procedure for cancers and also provides a material for genotyping, which can assist in the targeted therapies of cancers.

However, tissue biopsy-based cancer diagnostic procedures have limitations in their assessment of cancer development, prognosis and genotyping, due to tumour heterogeneity and evolution.

Studies found that the screening of genetic mutations using ctDNA is highly sensitive and specific, suggesting that ctDNA analysis may significantly improve current systems of tumour diagnosis, even facilitating early-stage detection.

Moreover, ctDNA analysis is capable of accurately determining the tumour progression, prognosis and assisting in targeted therapy. Therefore, using ctDNA as a liquid biopsy may herald a revolution for tumour management.

THE RESEARCH HISTORY OF ctDNA

It dates to year1977, when researchers made the novel observation that cancer patients carried cell-free DNA in their peripheral blood.

Indeed, significant progress was made with the advent of NGS technology (Next generation sequencing technology) in combination with the early findings of CGP (Comprehensive genomic profiling), which significantly improved the sensitivity and specificity of ctDNA detection.

(Next-generation sequencing (NGS) is a technology for determining the sequence of DNA or RNA to study genetic variation associated with diseases or other biological phenomena. Comprehensive genomic profiling (CGP) is a next-generation sequencing (NGS) approach that uses a single assay to assess hundreds of genes including relevant cancer biomarkers, as established in guidelines and clinical trials, for therapy guidance.)

Subsequently, research in this field has entered a “golden age” in which the huge potential of ctDNA investigations in tumour diagnosis and treatment is becoming ever clear.

THE BIOLOGY OF ctDNA

ctDNA is single- or double-stranded DNA and exists in plasma or serum. The mechanisms by which tumour DNA enters the bloodstream remain unclear.

It has been suggested that there are three potential origins of ctDNA

• Apoptotic tumour cells

• Living tumour cells

• Circulating tumour cells.

Generally, metastases represent the end-products of the invasion-metastasis cascade, which involves the development of the invasiveness capacity of cells in primary tumours, with subsequent blood dissemination of such cells and extravasation and metastasis to distant sites.

However, this theory has been challenged in the last decades because accumulating evidence has indicated ctDNA might play a key role in cancer metastasis through oncogenic transformation of susceptible cells.

METHODOLOGIES FOR DETECTION OF ctDNA-

Originally, researchers used Sanger sequencing to detect plasma ctDNA. However, there were many shortcomings for Sanger-based ctDNA detection, such as low-throughput, laborious protocols, high cost, and potential bias introduced by the PCR methodology. Diehl et al. developed a technique called BEAMing (beads, emulsion, amplification, and magnetics) to detect ctDNA in blood. Newman et al. developed another new technique called CAPP-seq (cancer personalized profiling by deep sequencing) for quantifying ctDNA. Compared with previous methods, the new techniques provide significantly higher sensitivity for ctDNA detection. Such “second generation” sequencing techniques have been essential in fully evaluating the clinical potential of ctDNA analysis.

The third-generation sequencing techniques are designed to be highly sensitive and inexpensive, have rapidly advanced and have the potential to expedite extensive application of ctDNA detection for routine patient management.

THE CRITICAL ROLE OF ctDNA IN CANCER DIAGNOSIS AND PROGNOSIS

Tissue biopsy is still the gold standard for tumour diagnosis. However, many shortcomings exist. There are risks associated with invasive sampling, particularly when applied to the fragile organs such as lung, and sensitivity is suboptimal, frequently resulting in an inability to detect early-stage tumours.

In addition, because tumours are heterogeneous and constantly evolving, tissue biopsy-based investigations are often unable to accurately determine tumour progression. Similarly, it is also difficult to detect small, residual lesions following therapy.

In recent years, it has been suggested that a plasma biomarker-based approach can evaluate the tumour occurrence, progression and recurrence. Such approach is minimally invasive with satisfactory conformity. However, previous tumour biomarkers in plasma only provide limited sensitivity and specificity, and therefore cannot always meet the clinical requirements.

ctDNA has been widely evaluated as a novel biomarker for liquid biopsy in cancer diagnosis and prognosis. Liquid biopsy based on ctDNA is superior to that of previous plasma biomarkers in sensitivity, and clinical correlations. Several studies have proved that ctDNA is more sensitive than protein biomarkers and CTC.

Importantly, the half-life of ctDNA is less than 2 hours, whereas the half-life of protein markers in plasma can be several weeks. This means that ctDNA can more accurately reflect the real-time tumour burden in patients receiving therapy.

THE CRITICAL ROLE OF ctDNA IN CANCER TREATMENT

In recent years, it has been suggested that genotyping has the potential to play a crucial role in precision medicine, especially in precision immunotherapy.

In current clinical practice, genotyping is achieved using DNA obtained from the tissue biopsy. However, tissue biopsy can only obtain local and static tumour information and is unable to reflect the real-time tumour genotyping due to heterogeneity and constant evolution of tumours.

ctDNA analysis overcomes these problems by reflecting the genetic mutations of the whole tumour tissue. Additionally, ctDNA from the same patients at different stages can be used to dynamically monitor the genetic mutations during the cancer progression.

Therefore, Liquid biopsy based on ctDNA analysis might improve tumour genotyping and targeted cancer therapy, which would be of significant benefit to the field of personalized medicine. Disease monitoring and treatment evaluation Cancer often relapses and evolves during the treatment. Disease monitoring and treatment evaluation are important for clinician to determine subsequent treatment protocols. It has been shown that serial ctDNA detection can be used to evaluate treatment efficacy by assessing remission status and detecting relapse and progression. Fast facts-

• ctDNA is fragmented DNA from tumour found in the blood.

• ctDNA testing is also called liquid biopsy.

• ctDNA can be isolated from blood and used to test for genetic biomarkers when tumour tissue is unavailable.

ctDNA is a promising biomarker in liquid biopsy for-

• Early detection of tumour

• Monitoring of tumour evolution

• Illustrate the mechanism of tumour resistance

• Guide in drug selection for the clinicians.

• Evaluate treatment efficacy by assessing remission status

• Detecting relapse and progression.

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