Clinical application of combined monitoring of cfDNA and CTC in the treatment of lung cancer

1. We have a biology laboratory and a PCR laboratory. 2. We have carried out a project on circulating tumor cells (CTC). 3. We have experienced, proficient, and qualified technical personnel. 1. We establish a predictive model for evaluating the therapeutic efficacy of different stages of lung cancer patients using CTC and cfDNA testing methods. 2. We establish standardized testing procedures and result interpretation standards. Evaluate the value of monitoring lung cancer treatment using both CTC and cfDNA detection methods individually and in combination. 1) Clarify the correlation between CTC and cfDNA detection and lung cancer treatment efficacy, and establish predictive models. 2) Publish 1-2 SCI papers. The subsequent achievements will serve as the preliminary foundation for continuing to apply for the project. The detection of cellular free DNA fragments (cfDNA) is an emerging and rapidly developing "liquid biopsy" technique in recent years. Research has found that there is an abnormal increase in cfDNA in the plasma and serum of cancer patients [1], indicating a correlation between cfDNA and tumors and potentially becoming a biomarker for evaluating prognosis and monitoring efficacy [2]. Up to now, cfDNA has been found to have characteristic gene changes in colorectal cancer, pancreatic cancer, lung cancer and other tumors, including point mutation, microsatellite instability, DNA hypermethylation, loss of heterozygosity, etc. [3]. Multiple studies have shown that the genetic changes in cfDNA are highly consistent with those in primary tumors and circulating tumor cells (CTC) [4,5]. The detection of CTCs or circulating tumor cell DNA (ctDNA) and cell-free DNA fragments (cfDNA) is an emerging and rapidly developing "liquid biopsy" technique in recent years. Research has found that there is an abnormal increase in cfDNA in the plasma and serum of cancer patients [1], indicating a correlation between cfDNA and tumors and potentially becoming a biomarker for evaluating prognosis and monitoring efficacy [2]. Up to now, cfDNA has been found to have characteristic gene changes in colorectal cancer, pancreatic cancer, lung cancer and other tumors, including point mutation, microsatellite instability, DNA hypermethylation, loss of heterozygosity, etc. [3]. Multiple studies have shown that the genetic changes in cfDNA are highly consistent with those in primary tumors and circulating tumor cells (CTC) [4,5]. CTC or circulating tumor cell DNA (ctDNA) can be used as a new biomarker for tumor diagnosis, treatment, and prognosis evaluation [6], but its content in patients' peripheral blood is low, making it difficult to extract and identify. Due to its complex operation, high cost, and long monitoring cycle, its practical application in clinical practice is limited [7]. Previous data indicates a strong positive correlation between cfDNA and ctDNA content [8]. Therefore, we can indirectly monitor the tumor's response to treatment by detecting the level of cfDNA. In addition, normal tissues or cells can also undergo apoptosis or necrosis after exposure to radiation, releasing a large amount of cfDNA in a short period of time, and the amount of change can indicate the risk of radiation damage [9]. How to detect changes in cfDNA content in blood with high sensitivity, accuracy, speed, convenience, and efficiency is the key to developing this "liquid biopsy technology" for clinical application. Our collaborative research unit has developed a nucleic acid signal amplification technology (SuperbDNATM), which indirectly reflects the overall level of cfDNA by detecting the free Alu sequence content in blood. The Alu sequence is a highly expressed repetitive sequence that accounts for approximately 10% of the genome [10] and is stable in whole blood [11]. Its expression is strongly correlated with the level of total cfDNA [12]. This technology platform has a sensitivity of 91.7% and specificity of 88.6% for quantifying cfDNA [13,14], and has obtained a global patent from the United States (patent number US2012/0003625A1). The feature of this technology platform is the use of modified BranchedDNA (bDNA) molecules and new probe design to enhance the amplification of labeled chemical signals on the target DNA sequence, without the need to amplify the target sequence itself (as shown in Figure 1). The working principle is as follows: Each oligonucleotide probe group contains two types of synthetic probes, namely capture extension probes (CEs) and labeled extension probes (LEs). Both CEs and LEs can bind to target DNA sequences. Firstly, MagPlex ™ Microspheres (magnetic beads) coupled capture probes capture target DNA sequences through co hybridization between CEs capture probes and CEs target DNA. Then, LEs can bind to both target DNA and pre amplified probes simultaneously. One pre amplification probe contains 20 sites that bind to the amplification probe, and one amplification probe also contains 20 sites that bind to the labeled probe. Therefore, the target signal was ultimately amplified 400 times. Finally, biotin labeled probes were combined with phycoerythrin labeled Streptavidin (SAPE), and the fluorescence intensity on the magnetic beads was measured by Luminex instruments such as MAGPIX. The fluorescence intensity is directly proportional to the number of DNA molecules present in the sample, and the level of cfDNA in the sample can be calculated. This method achieves highly sensitive quantitative detection of cfDNA concentration in plasma samples, with the advantages of no extraction, no amplification, easy operation, short detection cycle, and suitability for clinical testing needs. Canales and other scholars found through comparison that SuperbDNATM has certain advantages over PCR in detecting 244 common genes, such as smaller deviation, eliminating the impact of DNA extraction loss and amplification errors, avoiding sample cross contamination, and facilitating automation and batch detection [15]. This technology provides necessary technical support for monitoring cfDNA changes during tumor treatment.