[Introduction]ctDNA (circulating tumor DNA, ctDNA) is a DNA fragment that is usually actively secreted by tumor cells or released into the circulation during tumor cell apoptosis or necrosis. ctDNA carries genetic features derived from tumor cells, such as gene mutation, methylation, amplification or rearrangement, and can be used as an important indicator for tumor screening, companion diagnosis, treatment efficacy evaluation, and prognostic risk stratification. Because of its non-invasive or minimally invasive, short detection time, ability to reflect intratumoral and metastatic heterogeneity, and dynamic monitoring of therapeutic efficacy, it has been more and more widely used in the precise diagnosis and treatment of colorectal cancer (CRC). The main technical problems and quality control standards of ctDNA detection in practice need urgent attention.
In order to further standardize the application of ctDNA detection, the European Society for Medical Oncology (ESMO) Precision Medicine Working Group has invited a number of industry Well-known experts have comprehensively and systematically expounded the effectiveness and applicability of ctDNA testing in tumor patients, detailed the issues that should be considered when ctDNA testing is pre-processed, issued reports, and clinically used, and made relevant recommendations. . The article will be published in the ESMO official journal “Annals of Oncology” (Annals of Oncology) in July 2022.
This article will focus on the highlights of ctDNA detection technology, standardization of test reports and the value of ctDNA in the diagnosis and treatment of CRC for readers to exchange.
Technical Suggestions for ctDNA Detection
ctDNA detection is involved in many aspects of tumor diagnosis and treatment (Figure 1), including disease screening, postoperative minimal residual disease (MRD) assessment, recurrence prediction, advanced tumor genotyping, evaluation of treatment effects, response monitoring, and identification of resistance mechanisms. The detection methods of ctDNA include NGS sequencing, digital PCR, BEAMing and mass spectrometry, etc. The detection sensitivity and application scenarios of different platforms are also different. It should be noted that each detection method has its limitations. Therefore, for different scientific research or clinical problems, it is necessary to select an appropriate detection method and strictly follow the standards of each quality control link in the detection process, which is the key to the success of ctDNA detection. The impact of individual patient factors on ctDNA release, sample size, selection of collection tubes, sample storage and processing methods, etc., will affect the results of ctDNA testing to varying degrees. In this regard, the ESMO Precision Medicine Working Group has given the following 7 suggestions in terms of application.
Figure 1. Clinical application of ctDNA detection and ctDNA levels in different disease stages
The blood collection time for ctDNA testing should be carefully selected according to the clinical situation, because different factors can affect the release of ctDNA. The ESMO Precision Medicine Working Group recommends:
1) If postoperative detection of ctDNA-MRD is required, ideally at least 1 week after surgery; For more invasive procedures, testing needs to wait 2 weeks or more after surgery.
2) If the purpose of detection is advanced tumor genotyping, blood collection should be avoided from tumor patients with effective or non-progressive treatment to reduce false positives. Incidence of negative results.
Choice of blood collection tubes
Select according to the processing time of ctDNA samples and the detection method used (it is recommended to use anticoagulation tubes containing cell stabilizers, and as soon as possible complete plasma separation).
< span>DNA extraction and storage
Prior to ctDNA extraction, plasma samples should be stored at -80°C, while It is required that the temperature change be as small as possible, and the continuous freezing and thawing of the samples should be minimized.
False negative is a problem that cannot be ignored in ctDNA detection, which may be related to low plasma ctDNA levels, insufficient detection sensitivity or “non-existent” There are three factors related to non-shedding tumor (that is, the tumor itself does not release ctDNA).
CHIP (clonal hematopoiesis) is a common cause of false positives in ctDNA testing. Therefore, the ESMO Precision Medicine Working Group recommends simultaneous detection and analysis of plasma ctDNA and leukocyte DNA to identify CHIP mutations and reduce false positive rates.
Pathogenic germline mutations
ctDNA can detect pathogenic germline mutations in cancer susceptibility genes (eg, BRCA1, BRCA2, PALB2). In this case, the ESMO Precision Medicine Working Group recommends reflex germline testing using validated assays to differentiate between lineage and germline variation.
Clinical Genes Application of genotyping assays
By assessing tumor purity, clinical genotyping assays can improve the reliability of true-negative assay results.
Recommendations for standardizing clinical reports of ctDNA testing
In addition to the content contained in the routine clinical molecular laboratory reports, based on the ctDNA detection method and detection purpose, the ESMO Precision Medicine Working Group gave More comprehensive reporting recommendations (Table 1) further improve the standardization, accuracy, and practicality of ctDNA testing reports.
Table 1, what should be included in the ctDNA test report and report suggestions
ctDNA detection Application in the field of CRC
There are both commonalities and differences in molecular pathology between tumors with different origins or locations. With the deepening of molecular target research, ctDNA detection has also explored different applicable points in different tumors. In this ESMO Precision Medicine Working Group’s recommendations for ctDNA testing, we also focused on CRC-related content. (The content related to other tumor types can be obtained from the original link at the end of the article)
In terms of advanced CRC genotyping, ESMO Precision Medicine Working Group Commonly used molecular targets and related ctDNA detection suggestions are given (Table 2):
1) When tissue detection is not feasible or needs to be urgently done When making treatment decisions, ctDNA detection of KRAS/NRAS/BRAF V600E/MSI is recommended for chemotherapy-naïve metastatic CRC; span>
2) Before EGFR rechallenge, KRAS/NRAS/BRAF/ ctDNA detection of EGFR-ECD. It is worth mentioning that although KRAS mutations can occur in CHIP, the incidence is very low, so KRAS mutations in colorectal cancer patients are more likely to be true positive than false positive.
Table 2. Recommendations for ctDNA testing in advanced CRC genotyping
< span>In terms of dynamic monitoring of advanced CRC, a prospective study showed that after 2 cycles of first-line chemotherapy in patients with metastatic CRC, a 10-fold decrease in ctDNA was associated with progression-free survival (PFS). In another study, changes in ctDNA concentrations after 1 or 2 chemotherapy cycles predicted treatment response and PFS in patients with metastatic CRC[2-3]. Therefore, the ctDNA kinetic outcomes of patients with advanced CRC are closely related to patient prognosis. But there is currently insufficient evidence to show how action based on these findings could improve patient outcomes. Randomized interventional studies are still needed in the future to assess the clinical utility of ctDNA kinetic results.
In MRD/Molecular Relapse (MR) SurveillanceOn the other hand, a large number of studies have confirmed that the detection of ctDNA after treatment or during surveillance in early-stage CRC patients can effectively predict disease recurrence. At the same time, there are also a number of randomized controlled studies currently under development focusing on the clinical utility of ctDNA as a marker for CRC MRD/MR. However, the ctDNA detection method suitable for advanced tumor genotyping is not sensitive enough for MRD/MR, and the false positive rate caused by CHIP mutation is also high. Therefore, it is necessary to develop a targeted detection method to further utilize ctDNA detection in MRD/MR. The advantages and roles of monitoring.
In recent years, with the continuous development and success of clinical research on targeted and immunotherapy drugs, various The clinical treatment of tumor-like tumors has developed by leaps and bounds, and accurate detection and diagnosis have further promoted the precise treatment of clinical tumors. There is no doubt about the role of ctDNA detection in tumor diagnosis and treatment. With the continuous standardization and improvement of detection technology, detection methods, report interpretation and other aspects, ctDNA will also serve as a more convenient, more practical and more reliable liquid biopsy technology. clinical. In multiple links of CRC diagnosis, treatment, and follow-up monitoring, the application of ctDNA detection has its own particularity. Therefore, it is necessary to carry out targeted randomized controlled studies to explore the application of ctDNA in CRC. Better serve CRC patients with more evidence and confidence.
1. Pascual J, Attard G, Bidard FC, Curigliano G, De Mattos-Arruda L, Diehn M, Italiano A, Lindberg J, Merker JD, Montagut C, Normanno N, Pantel K, Pentheroudakis G, Popat S, Reis-Filho JS, Tie J, Seoane J, Tarazona N, Yoshino T, Turner NC. ESMO recommendations on the use of circulating tumor DNA assays for patients with cancer: a report from the ESMO Precision Medicine Working Group . Ann Oncol. 2022 Jun 9:S0923-7534(22)01721-5.
2. Tie J, Kinde I, Wang Y, et al. Circulating tumor DNA as an early marker of therapeutic response in patients with metastatic colorectal cancer. Ann Oncol. 2015;26:1715-1722.
3. Garlan F, Laurent-Puig P, Sefrioui D , et al. Early evaluation of circulating tumor DNA as marker of therapeutic effificacy in metastatic colorectal cancer patients (PLACOL study). Clin Cancer Res. 2017;23:5416-5425.
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