ctDNA applications and integration in colorectal cancer: an NCI Colon and Rectal-Anal Task Forces whitepaper

Abstract

An increasing number of studies are describing potential uses of circulating tumour DNA (ctDNA) in the care of patients with colorectal cancer. Owing to this rapidly developing area of research, the Colon and Rectal-Anal Task Forces of the United States National Cancer Institute convened a panel of multidisciplinary experts to summarize current data on the utility of ctDNA in the management of colorectal cancer and to provide guidance in promoting the efficient development and integration of this technology into clinical care. The panel focused on four key areas in which ctDNA has the potential to change clinical practice, including the detection of minimal residual disease, the management of patients with rectal cancer, monitoring responses to therapy, and tracking clonal dynamics in response to targeted therapies and other systemic treatments. The panel also provides general guidelines with relevance for ctDNA-related research efforts, irrespective of indication.

Fig. 1. Clinical applications of ctDNA.

Circulating tumour DNA (ctDNA) provides a more sensitive method of detecting malignancies than imaging or other conventional approaches. This sensitivity can be exploited in several ways: early diagnosis of colorectal cancer prior to the emergence of clinical or radiological manifestations and in the detection of minimal residual disease (MRD), defined as the detection of ctDNA with no other clinical evidence of disease recurrence in patients who have completed all potentially curative therapies. In patients with radiographically evident disease, ctDNA also seems to be more sensitive to changes in tumour burden and might assist in tailoring the intensity of therapy in the neoadjuvant setting and in monitoring for tumour response in patients requiring palliative treatment. Furthermore, qualitative assessments of the types of aberrations and their subsequent alterations might assist in assessments of tumour evolution and heterogeneity that lead to the emergence of resistance as well as in selection of the most appropriate therapies.

Fig. 2. ctDNA isolation and analysis.

a | Circulating cell-free DNA, of which circulating tumour DNA (ctDNA) is a part of, is isolated from plasma samples after serial centrifugation of blood collected in either K₂EDTA or cell-stabilizing tubes. ctDNA is subsequently isolated from cell-free DNA using library preparations and analysed for the presence of various genetic aberrations, including mutations, copy-number variations, fusions and/or other alterations such as changes in DNA methylation. b | Molecular barcoding: prior to PCR and sequencing, each DNA fragment can be labelled with unique DNA barcodes; subsequently, reads that share the same barcode (typically in thousands) can be grouped together because they all originate from the same ctDNA fragment. This approach enables sequencing errors (orange pentagon, seen in the minority) to be distinguished from true mutations (red pentagon, seen in the majority). Molecular barcoding also helps correct potential biases in amplification and thus assists in the precise quantification of mutations or amplification frequencies. NGS, next-generation sequencing; RBC, red blood cells.

Fig. 3. Applications of ctDNA in tailoring the aggressiveness of adjuvant therapy.

Owing to the high specificity of circulating tumour DNA (ctDNA) for the prediction of disease recurrence, patients with detectable ctDNA might be considered candidates for the escalation of adjuvant therapy over the standard-of-care approach in order to reduce the risk of disease recurrence. Conversely, patients who lack detectable ctDNA, as determined using a sufficiently sensitive assay, and who have a low risk of disease recurrence might benefit from de-escalation to less-intense adjuvant therapies that reduce the risk of toxicities.

Fig. 4. Tumour dynamics of patients receiving biomarker-selected targeted therapies.

Patients with RAS/RAF-wild-type, HER2-negative colorectal cancer receiving anti-EGFR antibodies demonstrate growth of clones of resistant cells over time, as reflected by increases in the variant allele frequency (VAF) of the resistance mutations present in these clones in circulating tumour DNA (ctDNA) (part a). Of note, the emergence of such aberrations in ctDNA typically predates radiographic or clinical disease progression. Such patients can be managed in several ways, including a ‘holiday’ from the targeted agent during the next line of therapy followed by the subsequent re-introduction of the original targeted agent based on a reduction in the VAF of resistance mutations on ctDNA-based monitoring (part b); by introducing a different biomarker-selected targeted therapy or an agent targeting treatment-emergent mechanisms of resistance, as determined by ctDNA findings (part c); or the introduction of one or more agents with activity against other downstream targets (part d).