ESMO Guideline on Liquid Biopsy Address Quality Standards for Clinical Implementation

In early-stage cancers, ctDNA has a role in screening, neoadjuvant monitoring, identification of molecular residual disease (MRD), molecular relapse monitoring, and early assessment of treatment response.

Recommendations for the use of liquid biopsies to detect circulating tumor DNA (ctDNA) were recently released by the European Society for Medical Oncology (ESMO). Investigators reviewed available evidence to provide tumor-specific and general consensus on the use of ctDNA and clarify quality standards required for decision-making.

The assay, which is minimally invasive and can provide more convenient and rapid results compared with tissue samples, has become increasingly used in clinical practice to molecularly profile tumors. The release of ctDNA is believed to be proportional to tumor growth with more ctDNA being shed as a tumor grows.

In early-stage cancers, ctDNA has a role in screening, neoadjuvant monitoring, identification of molecular residual disease (MRD), molecular relapse monitoring, and early assessment of treatment response. Of note, the guideline authors state there is a lack of clinical evidence to support the recommendation of ctDNA assays in these areas.

“New technologies currently in development, such as methylation pattern-based sequencing, fragmentation pattern-based sequencing or novel ultra-sensitive mutation detection methods, have the potential to optimize utility in these new settings,” the study authors wrote. “Multiple clinical trials are underway that may provide the evidence base to adopt ctDNA assays for decision making in multiple clinical scenarios.”

However, ctDNA assays have a larger role for patients with advanced disease as it can be used to guide adoptive adjuvant strategies upon clinical relapse, identify biomarkers to guide therapy and monitor response to therapy, and, upon disease progression, ctDNA assays can be used to identify acquired resistance mechanisms and the emergence of driver and resistance clones.

The evaluation of ctDNA varies and a single ctDNA assessment is fit for all purposes. Investigators noted that the ctDNA fraction changes based on tumor features such as extent of necrosis, disease burden, tumor location, inflammation, rates of proliferation and apoptosis, tumor microenvironment and host-related phenomena. Treatments such as chemotherapy, targeted therapy, radiation, and immunotherapy can also affect levels of ctDNA and ctDNA fraction.

Liquid genotyping consists of polymerase chain reaction (PCR)-based technologies and many studies have shown positive predictive values between ctDNA liquid biopsy and tissue-based PCR and next-generation sequencing testing in advanced malignancies (95%-99%). High accuracy of liquid biopsy in breast, gastrointestinal and lung cancer for single-nucleotide variants have been reported in large prospective studies of liquid biopsy.

The ESMO guidelines include tumor-specific recommendations for tier 1 and tier 2 variants across tumor types including the following:

Lung cancer: ctDNA genotyping recommended in treatment-naïve patients with cancer with resistance to prior TKIs. ctDNA testing may not have adequate sensitivity to detect MET true high copy number gain as resistance mechanism to osimertinib (Tagrisso) or lorlatinib (Lorbrena). Further, amplification and fusion detection is suboptimal with ctDNA assays, and should be repeated in tissue where possible.

Breast cancer:ESR1 mutations should preferentially be tested in ctDNA. HER2 amplification and NTRK fusions only when advanced tissue biopsy not available.

Gastric cancer and cholangiocarcinoma: ctDNA testing if tissue not available or when fast turnaround time is needed for urgent therapeutic decision-making.

Colorectal cancer:KRAS/NRAS/BRAF V600E/microsattelite instability for chemotherapy-naïve metastatic colorectal cancer is recommended when tissue testing is not feasible or urgent therapeutic decision-making. KRAS/NRAS/BRAF/EGFR-ECD for pretreated patients if EGFR rechallenge is planned.

Ovarian cancer: In women with no germline pathogenic BRCA1/2 variant found, testing for BRCA1/2 pathogenic or likely pathogenic somatic variants may be carried out if tissue not available.

Prostate cancer: BRCA1/BRCA2/ATM for potential PARP inhibitor therapy. Caution is needed when interpreting results of ctDNA assays due to false-positive CHIP mutations in DNA repair genes.

Pancreatic, hepatocellular, endometrial, urothelial, thyroid, and soft tissue sarcoma: ctDNA testing if tissue not available.

In terms of general tumor site recommendations, the ESMO guideline committee noted the occasions where tissue testing may provide confirmatory or more accurate guidance for treatment decision-making. These include interpretation of pathogenic variants such as BRCA1/2 or PALB2; retesing results that are deemed “not detected” by ctDNA; and for the detection of fusions and copy number events. Further, liquid biopsy should not be taken when a tumor is responding to treatment.

Liquid biopsy may have limitations for the detection of specific deviations, and they risk false negative results and the authors recommend that clinicians take these into consideration when evaluating the results. A false negative result may occur when a patient’s plasma sample does not have sufficient levels of ctDNA or ‘non-shedding’ happens, preventing the detection of a variant. False positive results may occur when clonal expansion takes place in apoptotic hematopoietic cells, where plasma DNA mainly originates; the frequency of clonal hematopoiesis of indeterminate potential (CHIP) increases with age, previous systemic cancer treatment and smoking. False positives can be minimized with either the sequencing of white blood cell DNA or the pairing sequencing of a tumor tissue sample. Additional limitations of ctDNA testing include a low tumor fraction presence in the sample, which prevents reliable assessment of variant allele fraction and limits copy number alteration analysis.

The guidelines recommend that clinicians consult a molecular tumor board for to assess results.


Pascual J, Attard G, Bidard FC, et al. ESMO recommendations on the use of circulating tumour DNA assays for patients with cancer: a report from the ESMO Precision Medicine Working Group. Ann Oncol. Published online July 6, 2022. doi:10.1016/j.annonc.2022.05.520