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Liquid biopsy analyses have redefined the constraints of cancer research, offering a noninvasive approach for the detection and monitoring of mutations critical to characterizing disease status. While pioneered primarily in the field of hematological oncology, liquid biopsy is a highly promising approach for monitoring changes in solid tumors. Assessments of circulating tumor DNA (ctDNA) in bodily fluids such as plasma and cerebrospinal fluid have shown to be valuable to clinicians and researchers for tracking cancer prognosis, progression, and relapse, while minimizing the number of costly and invasive procedures a patient must undergo.

The advent of ultrasensitive molecular tools, including next-generation sequencing (NGS) and Droplet Digital PCR (ddPCR) has made precise characterization of low-abundance biomarkers in liquid biopsies possible, and the latter enables absolute quantification of nucleic acid molecules. Clinicians are exploring these methods for accurate screening and molecular response monitoring, helping to assess both pre-adjuvant and primary treatment efficacy, and determine remission or relapse. However, the utility of ctDNA analysis research expands far beyond simple cancer detection and monitoring, informing precision treatment selection, uncovering drug resistance methods, and more. ctDNA analyses can offer deeper and more accessible insights into intra- and inter-tumor heterogeneity of solid tumors than traditional biopsy alone, driving continued progress in our understanding and treatment of cancer.

Ultrasensitive tools expand possibilities in ctDNA testing

While ctDNA can hold a wealth of information about cancer status, broadly used platforms, such as quantitative PCR (qPCR), limit the capabilities that investigators need for analysis. ctDNA analytes are often highly fragmented, present within a complex background of other biological components, and low in abundance, placing them below the limit of detection for qPCR. But highly sensitive molecular tools like NGS and ddPCR technology have enabled clinicians and researchers to access rich insights that ctDNA analysis can provide.

NGS testing has proven useful for conducting broad mutational analyses of tissue samples, screening for hundreds to thousands of mutations to provide a comprehensive picture of the genetic alterations present in a particular tumor tissue. ddPCR assays provide fast, ultrasensitive, and absolute quantification of target nucleic acids within ctDNA samples without the extensive bioinformatic burden associated with NGS testing. ddPCR technology can robustly detect subtle changes in nucleic acid targets, enabling precise assessment of low abundance ctDNA biomarkers. Along with its fast turnaround time and low cost, these features make ddPCR technology an ideal choice for serial sample analysis. However, a growing number of scientists are demonstrating the utility of highly sensitive ctDNA analysis in applications beyond treatment response and minimal residual disease (MRD) detection to forge new paths in precision oncology research.

Exploring new applications of sensitive ctDNA analysis

Precision Treatment Selection

Personalized cancer treatments targeting specific molecular pathways to inhibit tumor growth have yielded significant benefits in clinical outcomes and overall survival. While molecular testing of tumor tissue samples is still the gold standard for detecting and characterizing genetic alterations driving tumor growth, ctDNA analysis has emerged as an alternative or complementary approach in many instances. Particularly when time is limited or patients are medically unfit for repeat tumor biopsy, plasma-based ctDNA testing can help clinicians identify key mutations for targeted therapy selection.

A study of patients with non-small cell lung cancer (NSCLC) leveraged concurrent plasma-based and tissue NGS testing to detect targetable mutations.1 The addition of plasma testing nearly doubled the number of patients with targetable mutations detected when compared to tissue alone. Mutation-specific ddPCR assays have also demonstrated clinical utility in treatment selection. Researchers in the CHRONOS trial, a single-arm Phase II study of anti-EGFR therapy rechallenge for metastatic colorectal cancer, leveraged ddPCR technology to detect specific ctDNA mutations to guide second-line treatment selection after initial anti-EGFR therapy.2 Assays for acquired anti-EGFR resistance–conferring mutations in KRAS, NRAF, BRAF, and EGFR extracellular domains revealed mutations in 16 of 52 patients. Based on these findings, patients with wildtype RAS/RAF/EGFR were eligible for anti-EGFR rechallenge, presenting an alternative to standard late-line therapeutic options with limited efficacy and meaningful toxicities. In the absence of these mutations, anti-EGFR rechallenge has a response rate of 8–20% and more manageable toxicities than other available regimens. The use of ddPCR liquid biopsy in this setting filled an unmet need, providing molecular insights beyond the limits of challenging repeat tissue biopsy and offering patients an informed treatment alternative.

Accelerating time to treatment for advanced cancers

Particularly in advanced cases of cancer, wait times associated with molecular testing of tissue biopsies can significantly impact a patient’s time to treatment and overall outcome. Several studies have explored a “plasma-first” diagnostic approach to initiating treatment in patients suspected to have advanced lung cancer.

In one study of 49 patients with radiological evidence of advanced lung cancer, 11 were able to begin targeted treatment based on NGS molecular testing of plasma samples prior to receiving molecular testing results from tissue biopsies.3 The median time to results for patients who underwent plasma ctDNA analysis was 9 days, compared to 25 days for standard-of-care tissue tests. Another study of patients with suspected advanced lung cancer demonstrated that those who underwent “plasma-first” molecular profiling were able to begin treatment an average of one week sooner than patients who received only tissue biopsy genotyping.4 While tumor biopsy is ultimately required for diagnosis and subtyping of lung cancer, these trials have indicated that ctDNA molecular profiling prior to diagnostic tissue biopsy and profiling can help providers make timely treatment decisions, shortening patients’ time to treatment.

Characterizing drug resistance mechanisms

Detection of specific drug resistance markers can be vital for informing subsequent treatment selection in patients with advanced cancer. ctDNA-based methods have demonstrated clinical utility in identifying resistance markers, leading to the implementation of liquid biopsy in specific clinical settings. For example, patients with EGFR mutant NSCLC who develop resistance to first- or second-generation EGFR tyrosine kinase inhibitors can benefit from second-line treatment with osimertinib if an EGFR T790M resistant mutation is detected.5 A recent study also leveraged ctDNA analysis to identify acquired resistance-related alterations in metastatic colorectal cancer patients treated with anti-EGFR immunotherapy.6 These findings illustrate the value of ctDNA analysis as a complementary molecular diagnostic method for both initial and subsequent therapy selection in patients with advanced cancer.

Driving progress in oncology research with ctDNA analyses

Liquid biopsy analyses have emerged as a valuable, less invasive approach for assessing cancer prognosis, progression, and relapse. Advances in molecular tools have made ctDNA-based monitoring more accurate and precise but have also paved the way for the use of ctDNA analysis in other applications. Research demonstrating the utility of these analyses in precision treatment selection, characterization of drug resistance, and more has illustrated the massive potential of ctDNA molecular profiling as a complementary diagnostic method at every stage of cancer care. By leveraging ultrasensitive tools such as NGS and ddPCR technology, researchers and clinicians can continue to uncover new approaches to characterizing and treating cancer.



  1. Aggarwal C, Thompson JC, Black TA, et al. Clinical Implications of Plasma-Based Genotyping With the Delivery of Personalized Therapy in Metastatic Non–Small Cell Lung Cancer. JAMA Oncology. 2019;5(2):173–180.
  2. Sartore-Bianchi A, Pietrantonio F, Lonardi S, et al. Circulating tumor DNA to guide rechallenge with panitumumab in metastatic colorectal cancer: the phase 2 CHRONOS trial. Nature Medicine. 2022;28:1612–1618.
  3. Cui W, Milner-Watts C, McVeigh TP, et al. A pilot of blood-first diagnostic cell free DNA (cfDNA) next generation sequencing (NGS) in patients with suspected advanced lung cancer. Lung Cancer. 2022;165:34–42.
  4. Thompson J, Aggarwal C, Wong J, et al. OA16. 01 Plasma NGS At Time of Diagnostic Tissue Biopsy—Impact on Time to Treatment: Results From a Pilot Prospective Study. Journal of Thoracic Oncology. 2021;16(10):S876.
  5. Mok TS, Wu YL, Ahn MJ, et al. Osimertinib or platinum–pemetrexed in EGFR T790M–positive lung cancer. New England Journal of Medicine. 2017;376(7):629–40.
  6. Topham JT, O’Callaghan CJ, Feilotter H, et al. Circulating Tumor DNA Identifies Diverse Landscape of Acquired Resistance to Anti-Epidermal Growth Factor Receptor Therapy in Metastatic Colorectal Cancer. Journal of Clinical Oncology. 2023;41(3):485–496.


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