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Clinical Trials – What is holding liquid biopsy back?

This feature is written using content from our recently published Liquid Biopsy report, which you can read in full by downloading here.

Thanks to a wealth of publications demonstrating the exciting potential of liquid biopsy, the technique is now being evaluated in clinical trials to determine its true impact on oncological care. In this feature, we highlight major liquid biopsy trials in cancer, such as PATHFINDER and the NHS-Galleri study, and explore some of the limitations that are holding the technique back.

Currently, hundreds of trials are underway to analyse the clinical utility of liquid biopsy in various stages of the cancer treatment pathway1. From the early detection of cancer to monitoring disease recurrence, analysis of liquid biopsy biomarkers may potentially provide a rapid and non-invasive method to monitor cancer in all patients – regardless of cancer type or severity.

Despite impressive results, liquid biopsy is yet to be widely implemented within healthcare systems. With the high costs of liquid biopsy assays and many outstanding questions about their clinical utility, some are still unsure of the place of liquid biopsy within standard oncological care. However, large trials in diverse populations are now underway, possibly providing answers to those questions and driving liquid biopsy progress forwards.

Although the initial data from liquid biopsy trials looks promising, some limitations of the technique must also be recognised. Namely, the lack of standardisation and accessibility of the technology are prominent concerns amongst the liquid biopsy community. Until these issues are addressed, the widespread clinical implementation of liquid biopsy may remain challenging.

FIGURE 1: NUMBER OF CLINICAL TRIALS INVOLVING CFDNA ANALYSIS SINCE 2005. Sourced from Cisneros-Villanueva et al, 2022.

ctDNA trials making headlines

It’s clear that the field of liquid biopsy has exploded in recent years, with over 1,300 FDA and EMA cfDNA trials documented in 2022 alone1. Given the exponential nature of such trials (see Figure 1), this number is expected to have grown throughout 2023. It is therefore impossible to extensively document every liquid biopsy clinical trial. Instead, we present here a few notable highlights that have received significant media attention or published interesting findings in the last year.

Perhaps due to its well-established utility and detection protocols, cfDNA / ctDNA biomarkers are the target of most liquid biopsy clinical trials currently underway. This is also influenced by the development of NGS technologies, which offer higher accuracy and better resolution than ever before. Currently, liquid biopsy offers a necessary alternative when genomic profiling via tissue biopsy NGS is not feasible. To this end, many ctDNA trials have been established to validate liquid biopsy for tumour molecular profiling to guide treatment selection.

As a cancer with a highest incidence and second highest mortality rate worldwide4, lung cancer has received particular attention within the liquid biopsy community. In particular, 80 – 85% of lung cancer patients are diagnosed with non-small cell lung cancer5 (NSCLC), a common, slow-growing cancer that is prone to metastasis. Owing to its prevalence and relatively low survival rate, NSCLC trials are rapidly growing in number, with liquid biopsy being applied to various aspects of the NSCLC treatment pathway – including treatment allocation, treatment efficacy assessment, identification of drug resistance and MRD analysis2.

Reducing time-to-treatment for lung cancer patients is the primary goal of many clinical trials. In late 2023, we saw exciting results published from the accelerating lung Cancer diagnosis through liquid biopsy (ACCELERATE) trial, a nonrandomised study involving 150 individuals with suspected advanced NSCLC6. In this study, blood samples were collected from patients suspected to have NSCLC (based on abnormal imaging results) and subjected to NGS analysis. The liquid biopsy was performed alongside standard-of-care diagnostics, including regular tissue biopsy NGS profiling. Overall, plasma ctDNA testing was found to be significantly faster than other diagnosis methods, whilst also identifying actionable targets that were missed by tissue biopsy alone. However, NSCLC isn’t the only cancer in the spotlight of prominent liquid biopsy trials. In the last year, we saw a major liquid biopsy study established for patients with oestrogen receptor-positive (ER+) breast cancer. The trial, TRAK-ER, aims to detect molecular release earlier by identifying the presence of ctDNA after the initial treatment. By undergoing a liquid biopsy blood test every 3 months, investigators hope that patients at risk of relapse may have their treatment plans altered sooner, leading to better patient outcomes.

MCEDs trials

It is undeniable that one of the most exciting applications of liquid biopsy is in the early detection of cancer. Unsurprisingly, multi-cancer early detection (MCED) assays have risen to the forefront of the media coverage on liquid biopsy, generating an incredible amount of public interest. Several of these assays, such as GRAIL’s Galleri test and Exact Science’s Cancerguard test, are now the subject of extensive clinical studies to evaluate their effectiveness and specificity of cancer detection in real-world populations.

As the forerunner of MCED technology, the Galleri test is perhaps the most well-established and validated assay, with numerous clinical trials either completed or well underway. In late 2023, the results of GRAIL’s first major trial, PATHFINDER, were published7. Using an earlier version of the current Galleri assay, the trial followed approximately 6,600 participants for 1 year to analyse the performance of the MCED test as a diagnostic tool. Participants took the blood based Galleri test and their cfDNA was analysed, with those with a positive test result receiving further diagnostic testing informed by the Galleri cancer signal origin result.

Overall, the study was determined to support the feasibility of MCED testing in clinical practice. Importantly, the test can be used to screen for multiple cancers that currently do not have screening procedures in place, with the study determining that 74% of the Galleri-detected cancers were cancer types that do not currently have recommended screening tests.

Additionally, nearly half (48%) of participants that received a true-positive result had their cancers detected at an early stage (I – II), which could improve therapy options and patient outcomes. Although this data is promising, some concerns may be raised over the numbers of false-positive results – 62% of participants that received a positive result were confirmed to not have cancer after diagnostic assessment.

Building on this work, a second Galleri clinical trial is currently enrolling participants. The study, PATHFINDER 2, will investigate the performance of the current Galleri assay in approximately 35,000 North American participants. As in the PATHFINDER trial, those that receive a positive test will again receive targeted diagnostic procedures, with hopes that this MCED test may reduce the number and types of diagnostic procedures required to achieve diagnostic resolution. Moreover, this trial is planned to run for 3 years, lengthening the time for patient follow-up.

But no review of liquid biopsy trials would be complete without discussing the largest MCED trial ever conducted – the NHS-Galleri trial. The trial will offer 140,000 participants the opportunity to take the Galleri test once per year, for three years, with follow-up diagnostic tests and treatment if a positive result is generated. With participants being invited for their last test in July this year, it won’t be long until we see if the Galleri test will become a staple tool in the NHS’s long-term plan for early cancer detection. We spoke to trial leader, Charles Swanton, about the trial and gained some exciting insights into the process.

FLG: How might MCEDs change cancer screening within the NHS?

 Charles: Greater than 70% of cancer deaths right now occur in tumour types without an established screening programme. The potential benefit of MCEDs, if they are proven to be beneficial, is that they may be able to detect several cancer types for which there are no established screening programmes. There are promising data from some of the studies in this area already. Many MCEDs are based on blood tests that search for faint signals of cancer. It’s a complex area, as sensitivity is still limiting – they won’t be able to detect all cancers and specificity has to be maximized to limit false positives as much as possible. MCEDs may offer a potential opportunity for patients to access a screening methodology to detect some cancers that wouldn’t otherwise be detected.

FLG: Why was the Galleri test specifically selected for this trial, and what can you tell us about it?

Charles: GRAIL as a study sponsor has designed a large randomized controlled trial to evaluate their MCED test, Galleri. The trial needs to read out and the data assessed before the UK would adopt a programme like this. There have been several studies published with the GRAIL MCED test, including CCGA and the PATHFINDER program. On the basis of the early data, prior to trial initiation, the NHS approved a clinical trial programme to test its efficacy in real world populations.

FLG: What factors did you take into consideration when designing this trial?

Charles: Obviously, we had to target this study to a group of patients who were at risk of cancer, and this had to be a study done in asymptomatic populations. This had to be a large study, with the use of the NHS two-week wait pathway to investigate whether the patient with a positive test has an underlying occult cancer diagnosis. It used the NHS DigiTrials service that allows us to access patients across eight Cancer Alliance regions in England and approach them to take part in the study. In addition, we had to select an age group that was at risk of cancer. We know the risk of cancer increases dramatically after the age of 50, and this is a population of individuals who the test has been developed for in the US. Last but not least, we had to make sure that there was balanced recruitment across different ethnic and socio-economic groups, particularly targeting populations we consider to be at risk of cancer.

FLG: What metrics do you think are important in determining if the trial has been successful?

Charles: The trial aims to see if cancer detection can occur at earlier stages, shifting from late stage to early-stage disease. That’s the primary endpoint of the study. If it meets its primary endpoint, the study has been successful. In addition to that, Peter Sasieni and colleagues will be conducting analyses to look at cancer specific mortality over the next five or six years, to see if such a test has an impact on cancer-specific survival. This would be considered a gold standard for a screening trial. My belief is that stage shift is an important endpoint, because it gives the clinician an opportunity to intervene and escalate treatments in tumours at an earlier stage to increase the possibilities of cure. Studies powered for mortality would require many more patients and would not read out for 5 to 10 years. The first step is to assess whether the test can shift detection stage, from stage III – IV to stage I – II. We are hoping to see a readout in 2025. For now, that’s what I’m expecting.

Want to read the full interview? Download the report here.

Addressing the limitations of liquid biopsy

So, what is holding liquid biopsy back?

Despite numerous clinical trials demonstrating the potential benefits of liquid biopsy in the detection and monitoring of several cancers1, the technology is still far from widespread integration into clinical settings. Instead, liquid biopsies are often used as a complementary test alongside tissue biopsies8, perhaps due to several major limitations which are yet to be solved. One such issue, the lack of standard protocols, is a pressing concern that limits assay repeatability and comparison within clinical spaces.

Currently, there are no gold-standard protocols for liquid biopsy sample preparation (the pre-analytical stage) or downstream analysis and interpretation. This lack of standard protocols can lead to samples being collected too frequently that may result in resources being wasted9. Some argue that maintaining consistent practices in the preanalytical stage is of vital importance, as this ensures the integrity of the sample is not compromised. This reaches far beyond just liquid biopsy, with pre-analytical errors thought to comprise up to 75% of the total errors that occur in clinical laboratories processing blood-based tests10.

A number of variables could potentially affect the quality of the liquid biopsy data. In terms of pre-analytical steps, various aspects of the sample collection can differ between labs or assays, including: the type of collection tube used, time taken for sample processing, sample storage conditions and the sample processing protocol itself.

There are also a number of additional steps (such as the addition of sample stabilising reagents) where there are still no clear guidelines. Studies into some of the aforementioned variables have shown that the protocol selected significantly affects the amount of ctDNA detectable within the samples11. On a larger scale, researchers using varying protocols also affects clinical trials, making it harder to compare and collate liquid biopsy data between studies.

Unsurprisingly, the biological variation in ctDNA availability between different cancer types makes it difficult to develop a one-size-fits-all approach to liquid biopsy data interpretation and reporting. Additionally, cfDNA concentrations are known to fluctuate during the progression of the disease, or in response to treatment. Therefore, identifying appropriate standard timepoints for liquid biopsy testing will prove to be instrumental in the clinical adoption of the technique.

Given the importance of standardisation between assays, research groups and clinical trials worldwide, a number of organisations are working to bridge the gap and make uniform protocols a reality. Independent organisations and communities, such as BLOODPAC and the International Liquid Biopsy Standardization Alliance (ILSA), are working to develop reference standards and lexicons that can inform best practice for the wider liquid biopsy community.

Lastly, the ongoing clinical trials highlight a growing concern within the liquid biopsy field: who will get access to this groundbreaking technology? Within some universal healthcare systems (such as the NHS), liquid biopsy testing is expected to eventually be rolled out to all patients that require it over the coming years. However, in countries with limited healthcare options or where individuals must pay for medical services, the high cost of liquid biopsy testing may severely impact their accessibility.

Want to read more? Download the Liquid Biopsy Report here.


1. Cisneros-Villanueva, M. et al. Cell-free DNA analysis in current cancer clinical trials: a review. Br. J. Cancer 126, 391–400 (2022).

2. Parisi, C. et al. Circulating tumor DNA in clinical trials for solid tumors: Challenges and current applications. The Journal of Liquid Biopsy 1, 100007 (2023).

3. Sivapalan, L. et al. Liquid biopsy approaches to capture tumor evolution and clinical outcomes during cancer immunotherapy. J Immunother Cancer 11, (2023).

4. Casagrande, G. M. S., Silva, M. de O., Reis, R. M. & Leal, L. F. Liquid biopsy for lung cancer: Up-to-date and perspectives for screening programs. Int. J. Mol. Sci. 24, (2023).

5. Lung cancer statistics.

6. García-Pardo, M. et al. Association of circulating tumor DNA testing before tissue diagnosis with time to treatment among patients with suspected advanced lung cancer: The ACCELERATE nonrandomized clinical trial. JAMA Netw Open 6, e2325332 (2023).

7. Schrag, D. et al. Blood-based tests for multicancer early detection (PATHFINDER): a prospective cohort study. Lancet 402, 1251–1260 (2023).

8. Lone, S. N. et al. Liquid biopsy: a step closer to transform diagnosis, prognosis and future of cancer treatments. Mol. Cancer 21, 79 (2022).

9. Xie, W., Suryaprakash, S., Wu, C., Rodriguez, A. & Fraterman, S. Trends in the use of liquid biopsy in oncology. Nat. Rev. Drug Discov. 22, 612–613 (2023).

10. Green, S. F. The cost of poor blood specimen quality and errors in preanalytical processes. Clin. Biochem. 46, 1175–1179 (2013).

11. Crawford-Brown, C. The need for standardization in liquid biopsy sample prep. Today’s Clinical Lab https://www.clinicallab. com/trends/cancer-diagnostics/liquid-biopsy-sample-prep-lacksstandardization-22921 (2020).