Liquid biopsy in oncology: navigating technical hurdles and future transition for precision medicine

Hurdles of ctDNA analysis in liquid biopsy

Circulating tumor DNA (ctDNA), a subset of circulating cell-free DNA (cfDNA), originates from DNA fragments released during tumor cell necrosis or apoptosis. It carries tumor-associated genetic alterations, including somatic mutations, methylation changes, gene amplifications and chromosomal rearrangements. These molecular features enable ctDNA to serve as a critical biomarker for cancer screening, companion diagnostics, and real-time assessment of therapeutic efficacy in clinical oncology.

Meanwhile, the inherent shortcomings of ctDNA, including its current technical and biological constraints, could impede its broader clinical application. 1) due to its extremely low concentration in blood, mutation frequencies detected via ctDNA analysis are typically low, with artifactual signals often arising from ultra-deep sequencing amplification; 2) the release of ctDNA into circulation is influenced by tumor histology, anatomical location, disease stage, and tumor burden, all of which may further reduce ctDNA levels; 3) cfDNA derived from non-neoplastic cells interferes with ctDNA detection, complicating signal discrimination; 4) somatic mutations in cfDNA originating from clonal hematopoiesis can mimic tumor-derived mutations. Hence, all these interference factors lead to false-positive or false-negative results [2].

A more fundamental challenge lies in the biological origin of ctDNA which primarily reflects genomic alterations from dead tumor cells rather than actively proliferating ones [3]. Consequently, ctDNA-based next generation sequencing (NGS) may fail to capture emerging genetic heterogeneity within viable tumor cell populations which belongs to the minority new cancer cell clones, limiting its ability to monitor dynamic tumor evolution.

A more promising and applicable target for CTC analysis

Unlike ctDNA, CTC retains its intact cellular morphology and encompasses a complete set of genomic, transcriptomic, and proteomic information, making it the most accurate reflection of the true state of tumors. Consequently, NGS targeting CTC-DNA provides a more comprehensive representation of tumor heterogeneity.

However, the isolation and subsequent analysis of CTC present numerous challenges. Firstly, similar to ctDNA, CTC is present in very low abundance, making its acquisition difficult. Secondly, current CTC enrichment technologies have limitations. Comparatively, methods based on immunomagnetic bead enrichment theoretically offer better separation efficiency. The CellSearch platform, which targets the epithelial cell adhesion molecule (EpCAM) for CTC enrichment, is an automated CTC detection technology. It is the first CTC detection system to receive clinical validation and approval for use in the United States, Europe, and China [4], 5]. Its primary clinical applications include monitoring the progression of metastatic breast carcinoma (MBC), colorectal cancer, prostate cancer, lung cancer, and gastric cancer, as well as real-time assessment of patient prognosis and prediction of progression-free survival and overall survival. However, the number of CTC captured by this technology is extremely low, and interference from blood cells further complicates the process, making it currently insufficient to meet the demands of clinical usage for CTC detection. To improve CTC capture, the CellCollector detection platform was designed to coat EpCAM antibodies onto a metal wire, which is then placed in the median cubital vein for 30 min. As blood flows through the functional area of the wire, epithelial-derived tumor cells bind to the EpCAM antibodies and can be captured [6]. In addition, folate receptor (FR), cytokeratin, and epidermal growth factor receptor (EGFR) have also been reported as potential markers for CTC [7], 8].

It is worth noting that the enrichment of CTC based on these markers, regardless of EpCAM, FR, cytokeratin, or EGFR, is not tumor specific. During the process of tumor cells shedding from tissues into body fluids, epithelial-mesenchymal transition (EMT) often occurs, leading to a reduction or even loss of their expression levels. Consequently, the sensitivity and specificity of CTC capture in practice are suboptimal. Moreover, due to the presence of a large number of benign epithelial-derived cells in the body fluids (such as blood or pleural and peritoneal effusions) of cancer patients, reflecting that efficient and specific enrichment of CTC based on these markers cannot be achieved [9].

Utilizing monoclonal antibody library technology, Pan’s team discovered a novel tumor biomarker designated as tumor specific protein 70 (TSP70/SP70) with the relative molecular mass of 70kD [10]. Previous studies have demonstrated that SP70 is highly expressed on various cancer cell membrane, including lung cancer, gastric cancer, colorectal cancer, hepatocellular carcinoma, pancreatic cancer, breast cancer and ovarian carcinoma, while being completely absent in normal human blood cells. It was indicated that this new tumor biomarker could be used in the early diagnosis of various malignant diseases with different detecting technology [11], 12]. Given its features with high tumor specificity, SP70-targeted CTC enrichment has demonstrated high efficiency [13]. Therefore, the application of SP70-targeted CTC isolation technology will enhance the sensitivity and specificity of NGS detection with higher detection rate of hotspot gene mutations, compared to non-SP70-targeted clinical enrichment methods, potentially benefiting more patients and playing a significant role in the future of precision medicine.

Prospect

As mentioned above, ctDNA originates from dying tumor cells and inherently reflects the genetic background of low-viability tumor cell populations. One of the major challenges in liquid biopsy is to detect novel minority variant populations of cancer cells and corresponding mutations in apoptosis-resistant tumor cells, which are often under represented in ctDNA due to their survival advantage. In contrast, CTC exhibits stem cell-like properties and can detach from the primary tumor site, migrating into the bloodstream, lymph nodes or distant organs. Hence, CTC represents a more dynamic and viable tumor cell population, making it a superior source for genomic analysis. NGS targeting CTC-DNA holds significant promise for overcoming the limitations of ctDNA-based NGS. By capturing the genomic landscape of viable and metastatic tumor cells, CTC-DNA NGS can enhance the discovery and detection of tumor variants, including those associated with drug resistance and metastatic potential.

In addition, the consistency and comparability of detection results across different laboratories remain a major technical challenge in tumor sequencing applications. Due to variations in sequencing platforms, analytical software and workflows adopted by different facilities, significant discrepancies in outcomes are observed, which ultimately compromises the clinical utility of these results. The root cause of this issue lies in the absence of appropriate reference standards. The implementation of validated reference standards enables performance evaluation and platform calibration, ensuring that output data from diverse systems maintain consistency within defined parameters. This standardization is particularly critical for cross-platform data integration and comparative analyses in precision oncology research.

Future work ought to focus the identification of novel biomarkers with higher specificity and sensitivity for CTC isolation using monoclonal antibody libraries, phage display technologies, single cell proteomics, among others. Besides, due to the lack of standardization of ctDNA NGS and CTC-DNA NGS analysis, the Committee on Molecular Diagnostics in Oncology (C-MDO) of The International Federation of Clinical Chemistry and Laboratory Medicine (IFCC) is working at the standardization and optimization of liquid biopsy cancer testing. In conclusion, the shift from ctDNA to CTC-DNA analysis might represent a transformative approach in liquid biopsy, offering improved sensitivity and specificity for identifying tumor heterogeneity and actionable mutations. This advancement is critical for guiding precision oncology and developing targeted therapies against resistant and metastatic tumor cells.