Research progress of circulating tumor DNA in clinical application

Research progress of circulating tumor DNA in clinical application.  Molecular techniques can be very useful in detecting a patient’s tumor to guide treatment decisions is increasingly been applied in the care and management of cancer patients.

Circulating tumor DNA (ctDNA) containing mutations can be identified in the plasma of cancer patients during the course of the disease. As a non-invasive “liquid biopsies”,ctDNA is a potential surrogate for the entire tumor genome.

The use of ctDNA might help to determine the disease prognosis,monitor disease progression,monitor the molecular resistance and monitor the tumor heterogeneity. Future developments will need to provide clinical standards to validate the ctDNA as a clinical biomarker and improve the reproducibility and accuracy, in order to be better exploited for personalized medicine.

Cancer is one of the diseases with the highest morbidity and mortality in the world. In 2012, there were 14.1 million new cases and 8.2 million deaths worldwide. It is estimated that by 2035, the total number of cancers worldwide will exceed 24 million, and 14.6 million people will die from cancer [1].

There are various gene mutations and structural changes in the process of cancer. Detecting the types of mutations in tumor tissues can help understand the pathogenesis of tumors. If tumor-specific molecular markers are used for diagnosis, evaluation and prediction, good results can be obtained. Treatment effect [2].

One of the top ten breakthrough technologies of 2015 announced by MIT Technology Review-liquid biopsy, with its simplicity, sensitivity and specificity, non-invasive or minimally invasive, has brought a disruptive change to traditional cancer treatment. Among them, small fragments of free nucleic acid DNA in peripheral blood, circulating tumor DNA (ctDNA), as a potential tumor marker, have recently attracted much attention.

Discovery history and biological characteristics of ctDNA

In 1948, Mandel et al. [3] detected cell-free DNA (cfDNA) from normal human blood, but their pioneering work did not receive enough attention. In 1977, Leon et al. [4] found that the content of cfDNA in cancer patients was significantly higher than that of healthy individuals, while the content of cfDNA in patients with advanced cancer was higher.

With the continuous deepening of research, in 1989, Stroun [5] found that there are DNA fragments that are the same as tumor gene changes in the plasma and serum cfDNA of tumor patients, and scientists named it ctDNA [6]. Circulating DNA in the blood mainly comes from apoptosis, necrosis and secretion of cells [7]. Usually cfDNA is cleared in real time and maintains a very low content. There are only about a dozen nanograms (ng) of cfDNA per milliliter of plasma.

For example, in 10 ml of blood collected by conventional scientific research practices, there are about 20,000 cells of cfDNA. However, in tumor tissues, cell metabolism is vigorous, leading to a large number of apoptosis and necrosis, and increasing the release of free nucleic acid cfDNA. Therefore, fragmented, low-content ctDNA has the following unique characteristics:

(1) The fragment is short, about 150~200 bp[8];

(2) Short half-life: 15 min to several hours (about 2 h on average);

(3) The proportion of ctDNA in cfDNA ranges from 0.01% to 90.00%; (4) The level of ctDNA is related to tumor type, tumor burden and tumor progression [9].

ctDNA detection technology

At present, both quantitative analysis of ctDNA concentration and qualitative analysis of ctDNA can be performed, including the detection of gene mutations, deletions, insertions, fusions, rearrangements, copy number variations, methylation, and microsatellite instability (MSI) And loss of heterozygosity (loss of heterozygosity, LOH) and so on. Both quantitative and qualitative methods can reflect the existence and severity of tumors.

Common detection methods for ctDNA mutations and structural changes are roughly divided into two types, one is based on polymerase chain reaction (PCR) amplification, and the other is based on next generation sequencing (NGS) Detection. PCR-related technology has high detection accuracy and high sensitivity, but the information obtained is more limited, and can be used for trace nucleic acid detection; NGS-related technology has fast detection speed, wide coverage, and can achieve high-throughput detection of the whole genome, but the false positive rate needs to be further verification.

On this basis, scientists have developed a series of sensitive and specific ctDNA detection methods, including: quantitative polymerase chain reaction (qPCR), droplet digital PCR (droplet digital PCR, ddPCR)[10], digital PCR combined Streaming technology (beads, emulsion, amplification, magnetics, BEAMing) [11, 12, 13], Tagged-amplicon deep sequencing (TAm-seq) [14], Tumor personalized analysis deep sequencing method (cancer personalized profiling by deep sequencing, CAPP-Seq) [15, 16], whole genome sequencing [17, 18], whole exome sequencing, whole genome methylation sequencing, etc. [19]. Table 1 lists the comparison and application of some common ctDNA detection methods.

Clinical application of ctDNA detection

In traditional cancer treatment, doctors take out tumor samples through surgery or puncture needles, observe pathological tissue slices under a microscope and perform genetic analysis to make diagnosis and guide treatment. This method is invasive and risky, and it is relatively expensive. For the heterogeneity and drug resistance of tumor evolution, as well as the presence of multiple tumor foci in patients with metastasis, a single in situ biopsy has great limitations. ctDNA detection has the following advantages in many aspects: (1) Low invasiveness and non-invasiveness, minimizing the suffering of patients; (2) Sensitive and specific, high detection accuracy; (3) Easy sampling, multiple collections and real-time monitoring; (4) Comprehensive and extensive , Suitable for patients with tumor metastasis. Therefore, it has many applications in clinical medicine.

Early diagnosis The diagnosis of early patients with ctDNA detection technology is still in the stage of scientific research and exploration, especially for stage I patients, the sensitivity of detection is about 50% [15, 43]. Beaver et al. [28] used sensitive ddPCR detection to detect the hot spot mutation PIK3CA in tissues and plasma before surgery in 29 patients with early stage (stage Ⅰ ~ Ⅲ) breast cancer. 15 cases were in tumor tissues and 14 cases were in circulating blood. Mutations were detected in. The sensitivity of ctDNA detection was 93.3% and the specificity was 100%. Among the 10 patients whose plasma was detected with ctDNA before surgery, 5 patients still had trace PIK3CA mutations in the plasma after surgery. For early-stage patients, the detection of blood ctDNA for diagnosis is currently in the stage of scientific research and exploration, and can be used as a supplement to the preliminary screening of some hotspot mutations and tumor tissue detection.

Postoperative judgement The effect of the operation is evaluated by detecting the level of ctDNA, and it is judged whether the tumor has been removed. Surgery and chemotherapy can significantly affect the content of ctDNA. The concentration of ctDNA is significantly related to the survival rate of patients. The higher the concentration, the lower the survival rate of patients [13]. Sausen et al. [44] collected tumor specimens and blood samples from 101 patients with stage II pancreatic cancer, and performed whole-exome sequencing. The results found that in patients with early pancreatic cancer who underwent tumor resection, if ctDNA was not detected, the prognosis of successful surgery was good; if ctDNA was detected, there may be residual tissue that has a poor prognosis and is more likely to relapse. There are significant differences between the two groups of patients. . At the same time, compared with standard CT imaging, ctDNA can detect tumor recurrence 6.5 months earlier.

Table 1 Technologies for circulating tumor DNA (ctDNA) detection (reference)

Garcia-Murillas, a tumor molecular research team at the Cancer Institute of the United Kingdom, et al. [45] studied the changes of blood ctDNA in 55 patients with early breast cancer before and after surgery. All patients received surgery and chemotherapy. Using ddPCR and identification of somatic mutations in primary tumors, researchers found that ctDNA in blood samples after treatment was associated with a high risk of breast cancer recurrence. Among 15 relapsed patients, 12 (80%) had ctDNA detected in mutation tracking; and for patients who did not relapse, 96% of them could not find ctDNA in mutation tracking. After treatment, patients with positive blood ctDNA have a 12 times higher chance of cancer recurrence than patients with negative ctDNA. The ctDNA in the blood can detect breast cancer recurrence 7.9 months in advance.

Dynamic monitoring Using ctDNA as a tumor marker, multiple samples are taken to qualitatively and quantitatively detect tumor burden and monitor disease recurrence. At the same time, it can also track the response of the drug to the patient’s tumor, and take timely adjustment or treatment measures based on the drug efficacy information. When targeted drugs are effective, drug-sensitive tumor-specific mutations in ctDNA decrease, and once drug resistance develops, drug-resistant mutations in ctDNA increase.

At present, it has been reported in various cancers such as liver cancer [46], breast cancer [41, 45], pancreatic cancer [47], colorectal cancer [43], and non-small cell lung cancer [41]. One of the most classic cases is a 3-year follow-up study by researchers from the University of Cambridge on a breast cancer patient. Murtaza et al. [48] collected tumor samples and blood samples from a breast cancer patient whose tumor spread to other parts of the body, and carefully compared the ctDNA and biopsy collected at the same time point.

The results showed that the ctDNA in the blood samples matched the sequencing results of the live tumor samples, reflecting the same patterns and genetic changes as the tumor progressed and responded to drug treatment. Combined with the treatment plan taken by the patient, a series of plasma ctDNA mutation analysis was done during the treatment:

(1) Deep sequencing to observe the dynamic changes of PIK3CA mutation, PIK3CA mutation may be related to the tumor size during the use of tamoxifen and trastuzumab Related;

(2) Deep sequencing to observe the dynamic changes of ERBB4 mutation, ERBB4 may be the mutation site that causes lapatinib resistance.

It shows that ctDNA can monitor the disease in real time and assist in adjusting the treatment plan.

Medication guidance Through the detection of tumor-specific mutations in ctDNA, it can effectively reflect the patient’s response to treatment. Plasma samples before and after treatment were taken, and ctDNA sequencing was performed to identify drug-resistant mutations produced during drug treatment. Newman et al. [15] used the newly developed highly sensitive ctDNA detection method CAPP-Seq to detect early and advanced non-small cell lung cancer (NSCLC) patients. The results showed that a patient with stage Ⅵ NSCLC showed that the tumor shrank through imaging examination after 3 months of chemotherapy, and the ctDNA level decreased. However, 8 months later, the patient’s ctDNA level showed an increase, suggesting the progress of occult small tumor lesions.

The ALK fusion gene was found in the patient through ctDNA detection, and then the ALK fusion gene targeting drug crizotinib was used for treatment. The patient’s condition was greatly improved, and the ctDNA level also decreased again. In the treatment of NSCLC, the detection of epidermal growth factor receptor (EGFR) is very important to determine the treatment plan. Through plasma ctDNA detection, it can be found that when the new T790M mutation occurs, the patient is resistant to gefitinib[41], erlotinib or the combination of erlotinib and pertuzumab[49] . Once the T790M site mutation occurs, EGFR-tyrosine kinase inhibitor (EGFR-TKI) resistance will appear [50], and the treatment plan needs to be adjusted, and a new generation of irreversible EGFR-TKI inhibitors, chemotherapy, etc. Clinical options [51].

The presence of KRAS and BRAF gene mutations is the main cause of primary resistance to third-line treatment for metastatic colorectal cancer. Diaz et al. [52] tested the ctDNA of 28 patients receiving panitumumab for colon cancer and found that only 40% of wild-type KRAS Patients are sensitive to EGFR blockade therapy. Primary and acquired KRAS mutations during treatment are the reasons for insensitivity to EGFR blockade therapy. Combination therapy may be the most effective method for longer-term remission [53]. Detection of blood ctDNA for advanced colon cancer can effectively guide clinical medication [54].

Heterogeneous evaluation Cancer continues to divide in the patient’s body to produce new gene mutations, and drug resistance evolves so that cancer cells continue to survive and proliferate. ctDNA can be used as an effective marker to assess tumor heterogeneity. Garcia-Murillas et al. [45] of the Cancer Institute UK conducted ctDNA tracking on 55 patients with early-stage breast cancer who received surgery and chemotherapy as radical treatment. They found that in different individuals, the detected ctDNA mutations may only appear in the original. Tumors may occur only in metastatic tumors, and may also occur in two types of tumor tissues at the same time.

Murtaza et al. [48] used Bayesian clustering method PyClone to cluster the 207 functional mutations that have been discovered, and explored the following three groups of major tumor xenogenes: (1) mutations mainly occur in the early stages of cancer evolution; (2) mutations There is high abundance in all metastatic tumor samples, but it is not easy to find in the primary tumor; (3) Mutations are relatively diverse and are scattered in tumor biopsy samples at different stages. The random mutations contained in ctDNA in blood samples reflect the different sizes and activities of tumor monoclonal populations, revealing the sequence of tumor heterogeneity changes.

Other applications In addition to studying ctDNA in blood, scientists are also actively exploring the use of cerebrospinal fluid ctDNA as a marker for brain tumor liquid biopsy to overcome the influence of the blood-brain barrier [55], and there is a new report that ctDNA in the saliva of lung cancer patients has a detection effect similar to that of blood ctDNA[55] 56]. The American company Trovagene has persisted in researching urine ctDNA markers with abundant samples for many years. The ctDNA test has been recognized by some clinical and related institutions. The European Union and China successively approved AstraZeneca Iressa blood ctDNA companion diagnosis in 2014 and 2015, which is used to screen the patient population whose tumor tissue is not evaluable[57] .

Conclusion

Despite its great potential, ctDNA has not yet become the main clinical diagnosis and treatment method. The reasons are as follows:

(1) Early-stage cancer ctDNA detection is difficult [58]. The ctDNA detection technology CAPP-Seq can detect 10,000 blood DNAs for 1 tumor DNA, but even if the ultra-sensitive CAPP-Seq is used, the detection sensitivity of ctDNA is 100% in NSCLC patients at stage II to IV, while the detection sensitivity in stage I NSCLC is 100%. The sensitivity is only 50% [15]. Similarly, studies in nearly 10 types of 640 cancer patients also reported that 47% of stage I cancer patients, 55% of stage II cancer patients, 69% of stage III cancer patients, and 82% of stage IV cancer patients CtDNA was detected in the blood [43].

(2) The content of ctDNA in different tumor tissues varies greatly. Bettegowda et al. [43] found that less than 50% of medulloblastoma, metastatic kidney cancer, prostate cancer or thyroid cancer, and less than 10% of glioma patients can detect ctDNA; and in more than 75% CtDNA can be detected in advanced pancreatic cancer, ovarian cancer, colorectal cancer, bladder cancer, stomach cancer, breast cancer, melanoma, liver cancer, and head and neck cancer.

(3) The test results are uneven. ctDNA testing needs to strengthen the process specification, including the standardization of the blood treatment process and the extraction method of cfDNA, to improve the reproducibility of the test.

(4) The clinical cost is relatively expensive. Especially for the more effective and comprehensive second-generation sequencing technology, the price of ctDNA detection still needs to be further reduced before it may gradually become popular in clinical practice.

In summary, the detection of blood ctDNA has clinical advantages such as low invasiveness, non-invasiveness, sensitivity and specificity, real-time multiple times, and extensive comprehensiveness. Its clinical applications include postoperative judgment, dynamic monitoring, medication guidance, heterogeneous evaluation, and so on.

Although the ctDNA testing technology is not yet mature and a unified clinical standard needs to be established as soon as possible, with the increasing expansion of the depth and breadth of the basic experiment, clinical research and R&D application of ctDNA testing technology, the use of sensitive and specific non-invasive liquid biopsy to monitor cancer progress and follow-up Resistant mutations, timely guidance of medication plans, transformation of cancer from a deadly disease into a chronic disease, and precision medicine based on a customized model for each patient will no longer be a distant dream.

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