Many oncologists, including surgeons, medical oncologists, and radiation oncologists, have already started to incorporate ctDNA testing as part of their standard practice to monitor treatment response and aid in the interpretation of equivocal imaging findings and in surveillance for recurrence.
According to the American Society of Clinical Oncology (ASCO) and College of American Pathologists (CAP) in their review of the literature on circulating tumor DNA (ctDNA) in 2018, “There is no evidence of clinical utility and little evidence of clinical validity of ctDNA assays in early-stage cancer, treatment monitoring, or residual disease detection.”1
They then concluded that “given the rapid pace of research, re-evaluation of the literature will shortly be required, along with the development of tools and guidance for clinical practice.”
At the time of this joint statement, there wasn’t enough evidence to support the clinical utility of ctDNA for the management of cancer. However, the organizations were correct in their prediction that this was an emerging advancement in oncology that could change clinical practice. Since that statement, several prospective clinical studies have shown the clinical utility of ctDNA. Further, several companies, such as Natera, Inivata, Guardant Health, and Naveris, now provide CLIA (Clinical Laboratory Improvement Amendments)-grade ctDNA assays, which are starting to be used in routine clinical practice.
Circulating nucleic acids can be found in the blood, urine, and cerebrospinal fluid and the diagnostic assessment of them in liquid specimens is commonly referred to as liquid biopsies.2 Cell-free DNA (cfDNA), or circulating DNA, refers to extracellular fragments of DNA that are most often assessed in plasma specimens.
ctDNA is a subset of cfDNA that is derived from a patient’s tumor. Even in a patient with cancer, the ctDNA is only a small fraction of the cfDNA. The release of cfDNA from normal and cancerous cells occurs during cellular death, in which DNA fragmentation occurs by nucleases that cleave nucleosomal fragments. This fragmentation is nonrandom and mapping the ends of the cfDNA fragments reflects the nucleosomal profile of the cell it originated from. This may be used to distinguish between cfDNA arising from normal vs cancerous cells.3
Different cancers release different amounts of ctDNA; the amount of ctDNA released is also dependent upon the number of tumor cells that are in senescence vs undergoing apoptosis.4 Inhibiting senescence or activating apoptosis with radiation or chemotherapy can increase the amount of ctDNA released by cancer cells.
Typically, ctDNA is assessed in the plasma specimen from a peripheral blood sample, with the blood being collected in an EDTA tube or a cfDNA-stabilizing tube; moreover, ctDNA exists as DNA histone bundles which are relatively stable. At room temperature the DNA is stable for 4 to 6 hours in an EDTA tube and 5 to 7 days in a cfDNA-stabilizing tube. Once the plasma has been isolated via centrifugation, it is stored at –20 oC to –80 oC. Discriminating ctDNA from cfDNA is a technical challenge and there are various detection methods.5
ctDNA detection methods vary in sensitivity, and selection of the appropriate method depends upon the clinical situation, such as the patient’s tumor burden and the desired lower limit of detection. In general, digital droplet polymerase chain reaction and next-generation sequencing are the most used methods, but because of the high “background” noise of non–tumor derived cfDNA, some methods require whole exome sequencing of primary tumor tissue to develop a personalized ctDNA assay that tracks 16 to 20 mutations. Others require matched-paired peripheral blood cells.
Nonmalignant hematopoietic cells can accumulate clonal mutations, such as p53 mutations, which complicates the interpretation of cfDNA as mutations detected in plasma may not represent the true tumor genotype.6 ctDNA can be analyzed for the presence of various genetic aberrations, including mutations, copy-number variations, fusions, and/or other alterations, such as changes in DNA methylation.
ctDNA may provide clinical utility across the entire spectrum of cancer care.7 It may be useful in the early diagnosis of cancer in the screening setting, as it provides a more sensitive way of detecting malignancies than physical exams and imaging. Key to successful screening with ctDNA is screening patients who are at higher risk for developing cancer.
The high “background” noise of non–tumor derived cfDNA, especially clonal mutations from nonmalignant hematopoietic cells, will be a bigger issue in the screening space. Incorporating ctDNA analysis may also aide in the diagnosis of cancer in a patient in whom cancer is suspected. For example, circulating tumor human papillomavirus DNA (ctHPVD-NA) is detectable in more than 90% of patients with HPV-associated oropharyngeal cancer.8,9 The reported sensitivity and specificity of a composite noninvasive diagnostic using ctH- PVDNA and imaging or physical examination was 95.1% and 98.6%, respectively.9
Promising data exist regarding the use of ctDNA in the treatment and surveillance settings. Investigators of a prospective study in bladder cancer using the Signatera ctDNA assay (Natera) reported that detectable pretreatment ctDNA was prognostic and that postcystectomy ctDNA analysis correctly identified all patients with metastatic relapse during disease monitoring with 100% sensitivity and 98% specificity. The median lead time over radiographic imaging was 96 days.10 Similar findings have also been seen in studies of patients with breast, lung, and colon cancer.
The clearance kinetics of ctDNA during treatment may also be prognostic. Patients with HPV-associated oropharyngeal cancer who have rapid clearance of ctHPVDNA by week 4 of chemoradiotherapy may have better cancer control.8 In the surveillance setting, ctHPVDNA can detect tumor recurrence; moreover, data from a prospective biomarker study of patients with HPV-associated oropharyngeal cancer showed that ctHPVDNA post treatment surveillance had high positive predictive value and negative predictive value for identifying disease recurrence, with a median lead time between ctHPVDNA detection and biopsy-proven recurrence of 3.9 months.11 These results were validated with a commercial-grade ctHPVDNA test (NavDx, Naveris) done in a CLIA/CAP–certified laboratory in a 1076-patient prospective cohort study. The positive predictive value of ctHPVDNA surveillance was 95%, and ctHPVDNA positivity often preceded radiographic detection.12
Ongoing studies are now evaluating the tailoring of adjuvant treatment based on ctDNA. The intensity of adjuvant treatment, such as adjuvant radiation, chemotherapy, or immunotherapy, could be de-intensified, or even omitted, for patients who have undetectable ctDNA and intensified for those with detectable ctDNA after definitive treatment. Also, there is interest in conducting early intervention trials for patients who have early molecular relapse detected by ctDNA, with the hope that early salvage treatment will be more successful.
For patients with metastatic disease, ctDNA may be used to monitor response to treatment, especially when patients have equivocal imaging findings, and to identify targetable mutations to guide therapy selection when there is progression. ctDNA is commonly used in the management of non–small cell lung cancer (NSCLC) to evaluate for targetable gene mutations when there is insufficient tissue biopsy material for molecular testing.13
Targetable mutations may be better detected by ctDNA testing vs tissue testing because of increased sensitivity, the ease of obtaining a specimen, and inherent limitations of tissue testing. Guardant360 is the only FDA-approved ctDNA assay, and it is specifically for use in patients with stage IV NSCLC to detect targetable mutations.
Since the ASCO-CAP statement, a rapid growth in knowledge has taken place regarding the clinical usefulness of ctDNA in oncology. It has an established role today in NSCLC as an alternative to tissue-based analysis when selecting patients for targeted therapy, and there are still many other exciting potential applications and areas of investigation. Efforts should be made to make ctDNA testing cost effective, and in fact, in the surveillance setting it may be more cost effective than radio- graphic surveillance.14,15
Many oncologists, including surgeons, medical oncologists, and radiation oncologists, have already started to incorporate ctDNA testing as part of their standard practice to monitor treatment response and aid in the interpretation of equivocal imaging findings and in surveillance for recurrence. Thus, ctDNA is already in the clinic and oncologists have high hopes that it will help them personalize cancer care and improve the therapeutic ratio even more in the future of treating patients with cancer.
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 Clin Oncol. 2018;36(16):1631-1641. doi:10.1200/JCO.2017.76.867
2. Aucamp J, Bronkhorst AJ, Badenhorst CPS, Pretorius PJ. The diverse origins of circulating cell-free DNA in the human body: a critical re-evaluation of the literature. Biol Rev Camb Philos Soc. 2018;93(3):1649-1683. doi:10.1111/brv.12413
3. Snyder MW, Kircher M, Hill AJ, Daza RM, Shendure J. Cell-free DNA comprises an in vivo nucleosome footprint that informs its tissues-of-origin. Cell. 2016;164(1-2):57-68. doi:10.1016/j.cell.2015.11.050
4. Rostami A, Lambie M, Yu CW, Stambolic V, Waldron JN, Bratman SV. Senescence, necrosis, and apoptosis govern circulating cell-free DNA release kinetics. Cell Rep.2020;31(13):107830. doi:10.1016/j.celrep.2020.107830
5. Perakis S, Auer M, Belic J, Heitzer E. Advances in circulating tumor DNA analysis. Adv Clin Chem. 2017;80:73-153. doi:10.1016/bs.acc.2016.11.005
6. Hu Y, Ulrich BC, Supplee J, et al. False-positive plasma genotyping due to clonal hematopoiesis. Clin Cancer Res. 2018;24(18):4437-4443. doi:10.1158/1078-0432.CCR-18-0143
7. Dasari A, Morris VK, Allegra CJ, et al. ctDNA applications and integration in colorectal cancer: an NCI Colon and Rectal-Anal Task Forces whitepaper. Nat Rev Clin Oncol. 2020;17(12):757-770. doi:10.1038/s41571-020-0392-0
8. Chera BS, Kumar S, Beaty BT, et al. Rapid clearance profile of plasma circulating tumor HPV type 16 DNA during chemoradiotherapy correlates with disease control in HPV-associated oropharyngeal cancer. Clin Cancer Res. 2019;25(15):4682-4690. doi:10.1158/1078-0432.CCR-19-0211
9. Siravegna G, O'Boyle CJ, Varmeh S, et al. Cell-free HPV DNA provides an accurate and rapid diagnosis of HPV-associated head and neck cancer. Clin Cancer Res. 2022;28(4):719-727. doi:10.1158/1078-0432.CCR-21-3151
10. Christensen E, Birkenkamp-Demtroder K, Sethi H, et al. Early detection of metastatic relapse and monitoring of therapeutic efficacy by ultra-deep sequencing of plasma cell-free DNA in patients with urothelial bladder carcinoma. J Clin Oncol. 2019;37(18):1547-1557. doi:10.1200/JCO.18.02052
11. Chera BS, Kumar S, Shen C, et al. Plasma circulating tumor HPV DNA for the surveillance of cancer recurrencein HPV-associated oropharyngeal cancer. J Clin Oncol. 2020:JCO1902444. doi:10.1200/JCO.19.02444
12. Berger B, Hanna GJ, Posner M, et al. Detection of occult recurrence using circulating HPV tumor DNA among patients treated forHPV-driven oropharyngeal squamous cell carcinoma. Int J Radiation Oncology*Biology*Physics. 2022;112(5):e4. doi:10.1016/j.ijrobp.2021.12.016
13. Rolfo C, Mack P, Scagliotti GV, et al. Liquid biopsy for advanced NSCLC: a consensus statement from theInternational Association for the Study of Lung Cancer. J Thorac Oncol. 2021;16(10):1647-1662. doi:10.1016/j.jtho.2021.06.017
14. Kowalchuk RO, Kamdem Talom BC, Van Abel KM, Ma DM, Waddle MR, Routman DM. Estimated cost of circulating tumor DNA for posttreatment surveillance of human papillomavirus-associated oropharyngeal cancer.JAMA Netw Open. 2022;5(1):e2144783. doi:10.1001/jamanetworkopen.2021.44783
15. Ward MC, Miller JA, Walker GV, Moeller BJ, Koyfman SA, Shah C. The economic impact of circulating tumor-tissue modified HPV DNA for the post-treatment surveillance of HPV-driven oropharyngeal cancer: asimulation. Oral Oncol. 2022;126:105721. doi:10.1016/j.oraloncology.2022.105721