Liquid biopsies are quickly evolving beyond ctDNA, but technical challenges remain

Data from three presentations at the Molecular Analysis for Precision Oncology Congress (MAP) 2024 (London, 16–18 October) showcase the potential of circulating tumour cells (CTCs), exosomal microRNA (exo-miRNA) and cell-free RNA (cfRNA) from plasma samples to improve prediction of progression and early cancer detection, highlighting that the potential of liquid biopsy is not limited to the analysis of circulating tumour DNA (ctDNA).

The first study found significant reductions in the number of CTCs following 3 months of adjuvant treatment with CAPOX in patients with stage III colorectal cancer (Abstract 9P). In a prospective, non-randomised, single-centre trial, 175 patients received adjuvant chemotherapy with CAPOX or FOLFOX for 3 or 6 months. Around one half (47.9%) received 3 months of treatment (71.3% with CAPOX), while the other half received 6 months of treatment (65.5% with FOLFOX). Detectable CTCs were found in 45.5%, 40.7% and 50.8% of patients at baseline, after 3 months and after 6 months, respectively. Of note, significant reductions in the number of CTCs were seen after 3 months in patients treated with CAPOX (p=0.025). The researchers also observed significant CTC increases between 3 and 6 months in patients with right-sided tumours (p=0.022) and those aged less than 70 years (p=0.053). When circulating tumour DNA (ctDNA) was analysed by next-generation sequencing (NGS), detected ctDNA mutations were significantly associated with decreased disease-free survival and overall survival. CTC detection or increase during adjuvant therapy was not associated with recurrence, which occurred in one-third of patients at 4 years.

Commenting on these results, Dr Nicola Fusco from the European Institute of Oncology IRCCS, University of Milan, Italy, says, “Adding to investigations in metastatic disease (Crit Rev Oncol Hematol. 2024;203:104483), this interesting study suggests CTC detection may be useful in the adjuvant setting. There has been an increase in combination studies such as this, reflecting the recognition that CTCs and ctDNA can provide complementary information, making up for each other’s shortfalls.” He explains that ctDNA lacks the ability to capture the complex phenotype and biology of tumours, whereas CTCs provide a more holistic view and allow characterisation at multiple levels. As well as enumeration, isolated and enriched CTCs can be analysed using immunocytological, molecular or functional assays. The technology is now sufficiently developed that applications have been incorporated into clinical trials and Fusco thinks that even more opportunities will open up for CTCs, including in early relapse detection, treatment monitoring and the identification of resistance mechanisms, in a range of solid tumour types.

A second presentation described the development of an exo-miRNA panel for metastasis prediction in breast cancer (Abstract 17P). Exo-miRNAs were profiled from the plasma of 78 patients using NGS and analysed in comparison to data derived from ¹⁸F-fluorodeoxyglucose positron emission tomography/computed tomography used for staging. In total, 22 exo-miRNAs were identified that were differentially expressed in plasma from patients with early stage, advanced localised or metastatic disease. Fusco notes, “Compared with CTCs, using exo-miRNAs for staging and for predicting progression is still in a development phase and there are inevitable questions about reproducibility and clinical actionability. Nevertheless, this is an important proof-of-concept study and an area of growing interest, with many more exo-miRNA studies in progress.”

Finally, a poster detailed the current status of the development of a cfRNA-based multicancer early detection test (Abstract 48P). By combining NGS of cfRNA molecules with advanced machine learning techniques, the researchers aim to identify cancer-type specific biomarker signatures that can be detected at an early stage. So far, plasma samples have been analysed from 1,300 donors with colorectal, lung, breast, pancreatic or prostate cancer, non-cancerous diseases of the same organs or no disease. Biomarker signatures have been identified that are able to separate cancer from non-cancer patients. In addition, preliminary results indicate that the cfRNA sequencing process is sufficiently sensitive to identify patients with early-stage cancer or pre-cancerous conditions. “There is still a long way to go with this specific method and many technical challenges to overcome to develop a standardised, sensitive test that can be easily adopted,” says Fusco. “However, cfRNA, which encompasses diverse RNA types and captures pathogenic alterations in the transcriptome not detectable in the genome, is still well worth exploring in the important search for accurate early detection methods.”