Lung cancer is one of the malignancies with the highest incidence and mortality rates in the world, and seriously endangers human health [1]. Due to the lack of effective and accurate early diagnosis methods, many patients are diagnosed with advanced lung cancer and miss the optimal time for treatment, resulting in poor prognostic outcomes [2]. Traditional diagnostic methods include endoscopic ultrasound-guided fine needle aspiration (EUS-FNA), magnetic resonance imaging (MRI), low-dose spiral CT (LDCT), and histopathological diagnosis, but these methods have some drawbacks, such as invasiveness and radiation risks [3]. As a result, liquid biopsies are gaining attention due to their non-invasive and continuous sampling capabilities. At present, liquid biopsy biomarkers have achieved many results in the early diagnosis and prognosis assessment of lung cancer. In April 2024, the Journal of Experimental & Clinical Cancer Research published an online review titled "Liquid biopsy techniques and lung cancer: diagnosis, monitoring and evaluation", which aims to provide an overview of the molecular biomarkers and detection methods used in liquid biopsy of lung cancer. and elaborate its practical application [4]. This article focuses on the section "Biomarkers and detection methods for liquid biopsy of lung cancer" of the review.
Biomarkers and detection methods for liquid biopsy of lung cancer
At present, circulating tumor cells (CTCs), circulating tumor DNA (ctDNA), non-coding RNA, extracellular vesicles, tumor metabolites, tumor-associated antigens, and tumor-induced platelets have considerable potential as valuable biomarkers, promoting the application of liquid biopsy in the field of cancer research.
Circulating tumor cells
Circulating tumor cells (CTCs) are cells that originate from primary or metastatic tumor lesions and may enter the bloodstream as a result of spontaneous or medical procedures. The presence of CTCs greatly increases the risk of tumor metastasis and recurrence, as many of them are metastatic precursor cells. These cells are able to evade the body's immune clearance mechanisms and undergo epithelial-mesenchymal transition (EMT), resulting in enhanced mobility, adhesion, invasiveness, and penetration, promoting intravascular infiltration and distant metastasis.
Blood tests in CTC provide an important tool for the evaluation and management of tumors. By analyzing the morphology, genetic information, genotype, and heterogeneity of CTC, physicians can more effectively monitor patients' response to treatment, improve clinical outcomes, and provide patients with personalized and precise treatment plans. The clinical significance of CTC detection lies in its ability to be used for early diagnosis of tumors, prediction of metastasis and recurrence, assessment of risk of tumor progression, and real-time monitoring of the effect of drug therapy.
Detection techniques for CTC include immune enrichment and physical enrichment. Immune enrichment uses microfluidic chips or immunomagnetic bead technology to capture specific antigens on the surface of CTCs; Physical enrichment is based on the physical differences between CTCs and blood cells, such as size and density. The purpose of CTC enrichment is to reduce the interference of background blood cells and facilitate in-depth detection and analysis of CTC populations. Assays include protein expression analysis, immunocytochemistry, molecular nucleic acid detection, as well as histological imaging, tumor mutation analysis, and single-cell sequencing, which contribute to a comprehensive understanding of tumor heterogeneity and characterize CTC at different levels.
Although the exact relationship between CTC in the process of tumor proliferation and metastasis remains to be further studied, CTC provides valuable information for liquid biopsy and helps to fully understand the progression of the tumor. In the clinical treatment of lung cancer patients, CTC plays an indispensable role in early diagnosis, prognosis assessment and recurrence monitoring.
Circulating tumor DNA
Circulating tumor DNA (ctDNA) is a fragment of DNA that is released into the bloodstream after tumor cell death and is part of circulating cell-free DNA (cfDNA) and consists of the tumor cell's genetic code. ctDNA fragments are usually short, about 160 to 200 base pairs in length, and make up a small proportion of cfDNA, but are sufficient to provide direct evidence of the presence of a tumor. Compared to tumor tissue, ctDNA is less affected by tumor heterogeneity and has a shorter half-life of approximately 15 minutes to 2.5 hours, which allows it to serve as a real-time tumor marker to provide immediate monitoring of tumor dynamics.
The application of ctDNA detection technology has been approved by the U.S. Food and Drug Administration (FDA), and it has shown great potential in early diagnosis of tumor status, prognostic assessment, and monitoring of recurrence and metastasis. Testing of ctDNA focuses on finding genetic mutations and DNA methylations that may activate oncogenes or disrupt the function of tumor suppressor genes, thereby promoting tumor development. Due to the low levels of ctDNA in the blood, highly sensitive detection techniques such as amplification-refractory mutational systems (ARMS), quantitative PCR, digital PCR, and next-generation sequencing (NGS) are required.
In advanced or metastatic lung cancer, the detection of ctDNA is particularly important, not only to predict the effect of chemotherapy regimens, but also to assess the patient's response to treatment and survival outcomes. Specific mutations in ctDNA, such as EGFR mutations, can serve as molecular markers for tumor diagnosis and prognosis. In addition, ctDNA testing can help identify unknown tumor tissue variants in the case of chemotherapy resistance, providing an important tool for early diagnosis and prognostic assessment.
Liquid biopsy technology enables the detection of genetic mutations and the identification of new genetic changes associated with acquired drug resistance by collecting ctDNA from plasma from patients with non-small cell lung cancer (NSCLC). Customized treatment plans based on ctDNA test results can significantly improve the efficiency of clinical treatment. However, the detection of ctDNA also presents challenges, such as low ctDNA levels, short half-lives, and susceptibility to interference from other cfDNA, thus requiring highly sensitive detection methods. With the advancement of DNA sequencing technology, technologies such as ARMS, PCR, and NGS have made it possible to perform precise qualitative and quantitative analysis of ctDNA directly after DNA amplification. Nevertheless, the clinical application of ctDNA needs to be further validated and standardized through extensive clinical trials.
Non-coding RNA
Non-coding RNAs (ncRNAs) are a class of RNA molecules that play a key role in the regulation of gene expression, and they do not code for proteins, but can significantly affect gene and protein expression in cells. ncRNAs are mainly divided into small non-coding RNAs (sncRNAs) and long non-coding RNAs (lncRNAs) based on their length. sncRNAs are typically less than 200 nucleotides in length, while lncRNAs are more than 200 nucleotides.
ncRNAs are involved in a variety of biological processes during tumor development, including epithelial-mesenchymal transition, autophagy, and cellular senescence. In particular, microRNAs (miRNAs), as a type of ncRNA, are detected by liquid biopsy technology and show potential as early diagnosis and prognostic monitoring of lung cancer. Specific miRNAs, such as miR-125, miR-21-5p, miR-200b, and miR-141, are associated with lung cancer progression and chemosensitivity sensitivity.
Differences in the expression levels of lncRNAs, such as H19, MIR22HG, and LINC-PINT, in lung cancer tissues may make them biomarkers for lung cancer diagnosis and therapeutic targets. Circular RNA (circRNA) regulates gene expression by acting as a sponge of miRNAs, and its stability makes it a suitable biomarker for early diagnosis and recurrence monitoring of lung cancer.
At present, ncRNA research methods include transcriptome sequencing, real-time PCR, fluorescence in situ hybridization, RNA interference, and immunoprecipitation of RNA-binding proteins, which provide a variety of options for the detection of ncRNA and show great potential for application in the clinical screening of lung cancer.
The detection and analysis of non-coding RNAs is of great significance for understanding the complex properties and individual differences of tumors. With the advancement of liquid biopsy technology, ncRNA has broad application prospects as a biomarker in the early diagnosis, treatment response monitoring, prognosis evaluation and recurrence monitoring of lung cancer, providing new possibilities for precision medicine for lung cancer patients.
Extracellular vesicles
Extracellular vesicles (EVs) are nanoscale phospholipid bilayers secreted by cells, which play a key role in cell-to-cell communication and regulation of cellular activities. These vesicles are capable of carrying genetic material such as messenger RNA (mRNA), miRNA, lncRNA, nucleic acids, and proteins, which are essential for cellular function as mediators. EVs play an active role in the development and progression of tumors through cell-to-cell communication mechanisms, especially in the regulation of the immune microenvironment. As a subtype of EVs, exosomes have become a hot topic of research due to their stability and permeability.
EVs are seen as a promising alternative tool in liquid biopsy because they provide more accurate tumor information than circulating tumor DNA or necrotic circulating tumor cells. Genetic information such as miRNAs and lncRNAs carried by EVs play a crucial role in tumor progression, showing potential as biomarkers for early diagnosis of lung cancer.
However, EVs face some challenges in the development of liquid biopsies, mainly the low abundance of EVs in biological samples, which limits their isolation and characterization. Currently, the separation techniques used include ultracentrifugation, ultrafiltration, molecular exclusion chromatography, polymer precipitation, immunoaffinity chromatography, and microfluidics, each with its advantages and limitations. Microfluidic technology shows particular potential due to its portability, rapidity, low cost, ease of operation, and low contamination rate.
After EVs have been purified, their identification and characterization are critical to detect potential biomarkers carried. Exosomes are commonly identified by EV tracing, transmission electron microscopy, and particle size detection, while immunoblotting, ELISA, and flow cytometry are used to detect proteins carried by EVs. The development of new technologies such as electrochemical, colorimetry, and nanobiosensors has opened up additional possibilities for the identification and characterization of EVs and increased the sensitivity of detection.
Although EVs have shown promising biological properties in the diagnosis, prognosis, and treatment of lung cancer, there is an urgent need to optimize the isolation technology and improve its clinical translation efficiency to fully exploit the potential of EVs in liquid biopsy.
Tumor-induced platelets
During tumor tissue development, tumor cells are able to influence platelets through a variety of signaling molecules or receptors, leading to the formation of tumor-induced platelets (TEPs). These TEPs carry a wealth of spliced RNA biomarkers and RNA signatures, and have potential applications in tumor detection. Among them, tumor-derived platelet factor 4 has been shown to promote bone marrow megakaryocyte-mediated platelet production in NSCLC patients, and circulating platelets promote tumor progression by modulating tumor immune responses. In addition, platelets in the peripheral blood of NSCLC patients help identify clinically significant biomarkers and provide information about tumor spread and metastasis.
Techniques for the detection of platelet RNA include platelet RNA sequencing, microarray hybridization, and reverse transcription polymerase chain reaction, which enable platelet RNA to exert important oncologic diagnostic potential in blood liquid biopsy of lung cancer. RNA sequencing in NGS is valued for its ability to resolve multiple genetic information simultaneously. However, the complex isolation procedures of platelet RNA and the resolution of tumor markers from platelet RNA present challenges, and it is necessary to avoid interference and contamination by other biomarkers such as ctDNA and EVs in the process of isolating TEPs and resolving tumor-related markers.
In order to improve the efficiency of preclinical research, the platelet extraction and RNA sequencing process needs to be optimized and streamlined to reduce time and cost consumption. These efforts will help TEPs serve as biomarkers for liquid biopsy and provide valuable information for early diagnosis, treatment response monitoring, and prognosis assessment of lung cancer.
Metabolites
The growth and progression of tumors can induce physiological changes in the body, especially changes in the metabolic status of the whole body. During proliferation and metastasis, tumor cells adjust their metabolic pathways and release metabolites into the bloodstream. These metabolites can be used as tumor markers in liquid biopsy, which is helpful for early diagnosis, prognostic assessment, treatment response monitoring, and recurrence monitoring of lung cancer.
Glucose metabolism plays a key role in tumor cell proliferation, and its metabolites, such as 2-hydroxyglutaric acid, succinic acid, and transbutadielic acid, are important signaling molecules that may regulate tumor progression through epigenetic enzymes and DNA repair, and are associated with the survival rate and clinical prognosis of cancer patients. Lactate, as a major glycolytic metabolite, promotes tumor metastasis through histone modification, regulates gene expression and reprogramming of the tumor microenvironment. In addition, tumor lipid and amino acid metabolites, such as arachidonic acid and linoleic acid, also provide diagnostic possibilities, and amino acid metabolites such as tryptophan, leucine, and valine can be used as biomarkers to distinguish tumor and non-tumor patients.
Metabolomics techniques, including nuclear magnetic resonance (NMR) and mass spectrometry (MS), are essential for the comprehensive detection and accurate quantification of tumor metabolites. NMR is increasingly being used for metabolic fingerprinting studies and in vivo studies. MS techniques are generally divided into capillary electrophoresis-mass spectrometry, gas chromatography-mass spectrometry, and liquid chromatography-mass spectrometry. To improve the efficiency and accuracy of metabolite analysis, MS techniques are often used in conjunction with chromatographic separation techniques. Faced with the challenges of low levels of metabolites in the blood and complexity of sample preparation, the newly developed ion mobility spectrometry-mass spectrometry avoids assay overlap and expands the range of detectable metabolites. In addition, the strategy of acquiring both MS1 and MS2 spectra can improve the accuracy of identifying metabolic biomarkers.
The complex and dynamic changes of metabolites pose challenges to the application of metabolomics, and improved metabolomics methods and instrumentation are needed to overcome these challenges and address the current limitations in metabolite detection coverage, detection sensitivity, qualitative and quantitative accuracy, and tumor spatial information loss.
Tumor-associated antigens
Tumor-associated antigens (TAAs) are a class of molecular markers found on both tumor cells and normal cells, including embryonic proteins, glycoprotein antigens, and squamous cell antigens. Although TAAs are not endemic to tumor cells, they are produced in small amounts in normal cells and are highly expressed in actively dividing tumor cells. Aberrant expression of TAAs in the blood may contain valuable information about tumor activity, size, and genetic mutations.
The study found that there are autoantibodies against specific TAAs in the serum of lung cancer patients, which may indicate tumor proliferation, and therefore, TAAs have the potential to be used as biomarkers for the diagnosis of non-invasive early lung cancer. In particular, p53, a widely studied TAA, can affect the recruitment and activity of immune cells when deleted or mutated in tumors, allowing tumors to evade the immune system and promote cancer growth.
Current research relies on identifying known TAAs and testing the corresponding autoantibodies in patient serum. Key techniques include serological analysis of recombinant cDNA expression libraries (SEREX) and protein microarrays. The application of these techniques has yielded certain results, such as the isolation of 57 TAAs from the NSCLC-specific T7 phage library by the SEREX method, and the confirmation of their association with lung cancer progression by ELISA. Protein microarrays have also been used to identify specific TAAs associated with lung cancer, such as GAGE7, EEF1A, PMS2P7, NOLC1, and SEC15L2, which are significantly associated with different signaling pathways in lung cancer. These findings highlight the potential of TAAs in the early diagnosis of lung cancer and monitoring of treatment response.
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Disclaimer: This article is published with the support of AstraZeneca and is intended for healthcare professionals only
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