Diagnosis and Molecular Profiling

Isolation and analysis of ctDNA allows clinicians to identify genetic alterations, which may be used to guide treatment selection, depending on the cancer.1 Large, controlled studies suggest that there is up to 90% concordance of molecular profiling between ctDNA and tissue biopsy, provided that these measurements are collected simultaneously.

In 2016, the FDA approved the ctDNA assay cobas® EGFR Mutation Test v2 (Roche Diagnostics) for detection of EGFR mutations among patients with non-small cell lung cancer as a companion diagnostic to the EGFR inhibitor erlotinib.3 Presence of an EGFR mutation indicates that a patient is eligible for treatment with erlotinib or other EGFR inhibitors. Although this particular test can detect mutations in plasma, it can also be used to detect EGFR aberrations in tissue samples when tissue biopsies are possible.

A broader approach that uses NGS received the FDA’s breakthrough device designation in 2018.4 Using ctDNA, this assay evaluates at least 70 genes and other biomarkers, including microsatellite instability and blood tumor mutational burden, and could help guide treatment selection depending on cancer type.

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ctDNA could also be used for prognostication in this setting.5 The FDA approved a circulating tumor cell detection system, CellSearch, for patients with metastatic breast, prostate, or colorectal cancer. The amount of tumor cells present in the blood as measured by CellSearch is a predictor of survival; however, its use to justify a switch to more intense therapy has not been shown to prolong survival. Therefore, the CellSearch system has primarily been used for prognosis, rather than for therapy selection.

Monitoring Treatment Response and Resistance

Given the ease of collecting liquid biopsies, another attractive use of the tool is to evaluate the response to therapy by tracking ctDNA over time.1 For example, ctDNA levels initially increase after treatment, but then dramatically decrease within 1 to 2 weeks in patients who experience a response. Studies also suggest that ctDNA levels increase before progression can be detected radiographically, indicating that ctDNA may be useful in the future to help detect disease progression.

During monitoring after cancer treatment, ctDNA analysis may also detect the development of genetic alterations that result in treatment resistance. The FDA-approved cobas EGFR Mutation Test v2 can also detect the EGFR T790M mutation, which confers resistance to first- and second-generation EGFR inhibitors.

Residual Disease Detection

Patients with earlier-stage cancer who undergo resection with or without adjuvant therapy are at risk of relapse from any residual disease. A small study of 18 patients with colorectal cancer who underwent surgery demonstrated that ctDNA levels after resection was associated with relapse.6 Patients with no detectable ctDNA after resection did not experience relapse by their 1-year follow-up visit, whereas nearly all of the patients with detectable ctDNA relapsed within 1 year.

Cancer Screening

A major application for liquid biopsies is the early detection of cancer among seemingly healthy, asymptomatic individuals.1 The challenge is the potential for false positives. A study of more than 20,000 Chinese patients used Epstein-Barr virus DNA to screen for nasopharyngeal carcinoma.7 The liquid biopsy detected 309 individuals with EBV DNA, and 34 of those individuals were found to have nasopharyngeal carcinoma. One patient who was negative for EBV DNA was diagnosed with nasopharyngeal carcinoma a year later.

Another screening method is being developed, in which protein markers in cfDNA are detected by an assay called CancerSEEK.8 The assay was positive in a median of 70% of the 8 cancer types across 1005 patients with nonmetastatic but clinically detectable tumors. Among healthy controls, the specificity was 99%, indicating a very low false-positive rate—but sensitivities ranged from 69% to 98% for the detection of 5 cancer types (ovary, liver, stomach, pancreas, and esophagus). The addition, the use of supervised machine learning enabled tumor localization in 83% of cases. The input algorithm took into account the ctDNA and protein biomarker levels, as well as the gender, of each patient.

For the screening of colorectal cancer, the FDA approved of the Epi proColon® test, which determines the methylation status of the SEPT9 promoter among patients undergoing colorectal cancer screening.5 Although it is not a replacement for colonoscopy or sigmoidoscopy, the Epi proColon test was able to detect overall 71% of colon cancers, but was more sensitive to advanced disease.9,10 It offers an alternative to patients who refuse to or cannot undergo colonoscopy or sigmoidoscopy, with similar sensitivity and specificity as stool testing for advanced disease.10


Liquid biopsies, particularly those measuring ctDNA, offer an alternative to traditional tissue biopsy. Tissue biopsy, however, remains the gold standard, as ctDNA generally has not yet demonstrated clinical validity or utility in some cancers by way of large, randomized-controlled trials. In many cases, ctDNA is not recommended in lieu of tissue biopsy, but can be used if insufficient material is obtained by tissue biopsy, if there are contraindications to invasive biopsy, or if the tumor is inaccessible. As more studies validate the use of ctDNA, in the future, this approach may become a more equal alternative to tissue biopsy where histologic analysis is not also required.


  1. Corcoran RB, Chabner BA. Application of cell-free DNA analysis to cancer treatment. N Engl J Med. 2018;379(18):1754-1765.
  2. 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.
  3. United States Food and Drug Administration. FDA approves first blood test to detect gene mutation associated with non-small cell lung cancer [press release]. Published June 1, 2016. Accessed February 4, 2019.
  4. Foundation Medicine. Foundation Medicine’s new liquid biopsy assay granted breakthrough device designation by U.S. Food and Drug Administration. Press Release. April 26, 2018. http://investors.foundationmedicine.com/news-releases/ news-release-details/foundation-medicines-new-liquid-biopsy-assay-granted. Accessed February 4, 2019.
  5. Heitzer E, Haque IS, Roberts CES, Speicher MR. Current and future perspectives of liquid biopsies in genomics-driven oncology. Nat Rev. 2019;20:71-88.
  6. Diehl F, Schmidt K, Choti MA, et al. Circulating mutant DNA to assess tumor dynamics. Nat Med. 2008;14(9):985-990.
  7. Allen Chan KC, Woo JKS, King A, et al. Analysis of plasma Epstein–Barr virus DNA to screen for nasopharyngeal cancer. N Engl J Med. 2017;377:513-522.
  8. Cohen JD, Li L, Wang Y, et al. Detection and localization of surgically resectable cancers with a multi-analyte blood test. Science. 2018;359(6378):926-930.
  9. Nian J, Sun X, Ming SY, et al. Diagnostic accuracy of methylated SEPT9 for blood-based colorectal cancer detection: a systematic review and meta-analysis. Clin Transl Gastroenterol. 2017;8(1):e216.
  10. Final Recommendation Statement: Colorectal Cancer: Screening. United States Preventive Services Task Force. June 2017. https://www.uspreventiveservicestaskforce.org/Page/Document/ RecommendationStatementFinal/colorectal-cancer-screening2. Accessed February 4, 2019.