Summary of research progress in circulating tumor DNA (ctDNA) detection
Release date: 2018-04-18
Recently, the American Society of Clinical Oncology (ASCO) and the American Association of Pathologists (CAP) organized a panel of experts to conduct a comprehensive review of published clinical ctDNA testing papers, which comprehensively summarized ctDNA (circulating tumor DNA). Detecting current research status and issues that need further resolution also provides framework recommendations for future research, better guiding and standardizing the development and application of ctDNA detection technology.
First, the background introduction
Liquid biopsy technology refers to the diagnosis and analysis of blood and other specimens of body fluids. It has the advantages of convenient sampling, minimally invasive, easy to achieve dynamic monitoring, more complete reaction of tumor gene information, and overcoming the spatial limitation of single lesion sampling. Among them, the detection of blood ctDNA is currently the fastest growing detection method in the field of liquid biopsy.
A total of 1338 clinical literatures related to ctDNA detection were searched by literature search, and 77 articles were finally screened for analysis. The main contents of the ctDNA mutation and DNA sequence or copy number variation in solid tumors are analyzed from the aspects of sample pretreatment, analysis effectiveness, interpretation and report of results, clinical validity and practicability. The main contents are as follows:
Second, sample pretreatment
There are many uncertain factors in the pre-processing steps of ctDNA detection samples, and these variables may affect sample quality, DNA extraction, etc., thus affecting subsequent detection and evaluation.
Current research indicates that the best sample type is plasma, and blood is preferably collected using an anticoagulation tube containing an cell stabilizer or an EDTA anticoagulation tube. When an EDTA anticoagulation tube is used, it should be treated as soon as possible within 6 hours after sampling to separate the plasma. The dilution effect of a large amount of free DNA produced by leukocyte lysis on ctDNA should be minimized in this process.
In general, there are many factors that may affect subsequent testing in the steps of sample blood collection, collection, processing, storage, transportation, DNA extraction and purification. In addition, patient-related biological factors may also affect the release of free DNA in the patient's blood, such as smoking, anemia, inflammatory non-malignant diseases, autoimmune diseases. At present, research in this area is still limited, and many problems remain to be resolved.
Third, the effectiveness of the analysis
Analytical effectiveness refers to the ability to accurately and reliably detect mutations, including accuracy, sensitivity, specificity, and reproducibility. At present, the method for evaluating the accuracy and specificity of plasma ctDNA detection is mainly by methodological comparison with paired tissue sample detection. Studies have shown that there is an inconsistency between plasma ctDNA detection and tissue detection. This situation may be caused by other analytical factors, such as cancer type, tumor stage, tumor space and time. Qualitative, tissue plasma sampling time difference, etc.
Due to the low content of ctDNA in plasma, it is important to study the lower limit of detection. Considering the possible influence of the above biological factors, it is recommended to include cell lines or artificially constructed standardized samples to verify the detection ability near the detection limit. At present, the optimal detection limit for different mutation types has not been systematically studied, and the detection limit may also vary depending on the intended use.
In addition, there are differences in current ctDNA detection capabilities, and future studies should consider more large cross-platform alignment studies. At the same time, the next step should be to consider the variables in the sample pre-processing and analysis and testing to ensure the quality of the test. Finally, it is recommended that the lowest analytical sensitivity and specificity of ctDNA assays be established based on the intended use to facilitate their clinical application.
Fourth, the results of interpretation and report
The interpretation of ctDNA test results faces some unique challenges, especially the following points:
(1) When the ctDNA test report shows that a specific gene mutation can be treated, it is recommended to integrate the test results with other clinical information and tumor analysis information to illustrate the overall correlation with the treatment plan. In the application of detectable germline mutations, attention should be paid to subsequent detection and interpretation of ambiguous results, or possibly germline mutations.
(2) Gene mutations in clonal hematopoiesis of unknown significance should be interpreted with caution.
(3) The ratio of ctDNA to total cell free DNA in different patients is quite different. The free DNA released by leukocytes in samples obtained from different pretreatments is not the same. The prognostic significance of gene mutation abundance in ctDNA detection is needed. .
(4) ctDNA test results may be inconsistent with tissue test results, considering that the release of ctDNA in plasma is usually low, especially in the central nervous system cancer, the blood-brain barrier may have a greater impact, so for undetected results Carefully interpret and explain.
V. Clinical effectiveness and practicality
According to the different expected uses of ctDNA detection, the article focuses on the following aspects, mainly focusing on the application in metastatic cancer.
(1) Selection of treatment options for advanced cancer
There are relatively many studies on the application of ctDNA in this area, and there are currently approved products based on PCR to detect EGFR mutations in patients with non-small cell lung cancer. Its clinical applicability is mainly obtained through retrospective analysis of samples obtained in previous clinical trials. Among them, it is necessary to pay special attention to the fact that due to the existence of partial false negative results, especially in the case of T790 detection, the results of undetected should be confirmed by subsequent tissue testing as much as possible.
In addition, due to the presence of tumor heterogeneity in advanced cancer, ctDNA may be from all metastatic sites, and tissue testing is usually performed at a single metastatic site or primary lesion. High-sensitivity ctDNA detection may detect subclonal mutations, and in different cases, the possible effects of subcloning mutations deserve further study.
On the whole, except for the tests that have been approved by the regulatory authorities, most of the testing methods do not have sufficient evidence to show their clinical effectiveness, and most of them do not have clinical practical evidence. The next study should be included as close as possible to the expected population. The patient and the sample should undergo strict quality control and carry out prospective clinical studies to fully verify its clinical significance.
(B) applied to efficacy monitoring
When ctDNA is used for therapeutic monitoring, it is mainly to quantitatively detect changes in ctDNA over time. However, there are still many challenges in this process, including considering the effectiveness of sample preparation and analysis processes and the impact of repeatability. Research is needed to verify the interchangeability between different laboratory results and establish the best unit for quantifying DNA load. Wait.
Although the relationship between ctDNA levels and treatment feedback has been established in a small number of studies, there is currently insufficient evidence for clinical efficacy of this application, and there is a lack of strong clinical practical evidence that requires further rigorously designed clinical studies.
(3) Application to the detection of residual lesions
Although in theory, after patients undergo radical treatment such as surgery, residual tumor tissue can be detected according to changes in ctDNA to diagnose or assist in judging cancer recurrence at an early stage. However, there is no evidence that ctDNA can achieve the detection of small residual lesions similar to leukemia management, and most of the research in this area is retrospective. The false negative rate and false positive rate of the test have not been determined, and the clinical validity evidence Limited, clinical applicability is not yet clear, and it is recommended to conduct prospective randomized studies in the future.
(4) Tumor screening for asymptomatic people
Considering that ctDNA can be detected in early cancer patients, but its content is usually low, the use of this test for cancer screening in asymptomatic populations has received much attention. However, there is still no relevant clinical validity and clinical practical evidence for the field of solid tumors. The extent of detecting false positives (including technical and biological factors) is still unclear.
Conclusion
Although ctDNA detection has shown many advantages in the field of tumor diagnosis, there are still many problems to be studied in terms of sample pretreatment, analytical performance, clinical effectiveness and clinical applicability, especially in clinical efficacy and clinical practical evidence. In addition, in addition to the individual approved products to some extent indicate their clinical use, most of the tests used in cancer screening, treatment options, efficacy monitoring, residual disease detection, lack of strong clinical evidence, at this stage is not appropriate In clinical practice, the next study should include all possible expected population types as possible, and fully validate its clinical significance through rigorously designed clinical studies.
references:
[1] Merker JD, Oxnard GR, Compton C, et al. Circulating Tumor DNA Analysis in Patients With Cancer: American Society of Clinical Oncology and College of American Pathologists Joint Review [J]. Journal of clinical oncology, 2018: JCO2017768671.
[2] Oxnard GR, Thress KS, Alden RS, et al. Association between plasma genotyping and outcomes of treatment with osimertinib (AZD9291) in advanced non-small-cell lung cancer[J]. Journal of clinical oncology, 2016, 34( 28): 3375.
[3] Wu Yilong, et al. Liquid biopsy: standard and precise peers [J]. Evidence-based medicine, 2016, 16 (4): 193-197.
[4] Liu Yi, Zhang Shaohua, Wu Yilong, et al. Progress in clinical oncology in China [M]. People's Medical Publishing House, 2017: 435-437.
Source: China's review
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