Introduction

The past decades have witnessed an explosion in therapeutic options for both early stage and advanced-stage malignancies. This increase in available therapies has been accompanied by growing interest in develo** more personalized treatment approaches, thus increasing the demand for molecular profiling strategies that enable the successful delivery of precision medicine.

The term ‘liquid biopsies’ refers to biomarkers found within biological fluids, traditionally blood, that can be sampled to provide clinically valuable information on both the patient and their underlying malignancy1. This approach offers an alternative to traditional needle-based biopsy sampling methods that enables minimally invasive repeat biopsy sampling, even for patients with tumours that are anatomically difficult to sample directly. Liquid biopsy approaches can also overcome issues such as a lack of representativeness, which is associated with the analysis of small tissue biopsy samples2. To date, the majority of liquid biopsy research has focused on blood-based biomarkers, including circulating tumour DNA (ctDNA), circulating tumour cells (CTCs) and circulating microRNAs3. However, data published over the past decades indicate that these cancer-derived cells and molecules can also be found within non-blood biological fluids, including, among others, cerebrospinal fluid (CSF), urine, pleural fluid and peritoneal fluid4,5,6,7,8,9,10. This expanding interest in liquid biopsies from a wider range of sources is supported by the improved availability of ctDNA detection and analysis technologies. In this Review, we discuss the role of non-blood biological fluids as sources of ctDNA and their potentially distinct and/or complementary roles in oncology (Box 1) as well as exploring some of the challenges associated with translating these alternative biomarker assays from the laboratory to the clinic.

Definitions and assay considerations

The term cell-free DNA (cfDNA) refers to fragments of DNA that are present outside of cells that can be detected within bodily fluids. In blood plasma, cfDNA typically consists of double-stranded DNA fragments of around 140–170 base pairs (bp) in length that mostly originate from leukocytes11,12. ctDNA refers to the portion of cfDNA derived from cancer cells, which typically comprises strands of <145 bp in length and is responsible for the substantially higher plasma cfDNA concentrations often seen in patients with cancer11,13,14,15 (Box 2). Various studies have confirmed a high level of concordance between genomic alterations detected in plasma ctDNA and those found in tumour tissues16,17. Nonetheless, the mechanisms of ctDNA release are still poorly defined and various hypotheses include tumour cell apoptosis, direct release from tumour cells, secretion from macrophages following phagocytosis of tumour cells and, to a lesser degree, release from CTCs13.

ctDNA can also arise from direct contact with tumour tissues in non-blood biological fluids, and these fluids might therefore contain a higher proportion of ctDNA than plasma. Furthermore, the comparative absence of cfDNA originating from haematopoietic cells in non-blood sources means that ctDNA might be present at higher relative concentrations and at higher variant allele frequencies (VAFs) compared with ctDNA isolated from blood18. This relative lack of cfDNA can be advantageous when quantifying ctDNA and when attempting to detect alterations present at low relative frequencies owing to a lack of the background noise created by clonal haematopoiesis, which typically increases with advancing age and/or previous radiotherapy19. Furthermore, ctDNA from non-blood sources might be more reflective of the primary tumour (as seen in pleural fluid samples obtained from patients with advanced-stage thoracic malignancies) or, alternatively, might enable the characterization of a genetically distinct population of tumour cells, such as those located in the CSF in patients with intracranial disease18,20.

A variety of assay types can be used to measure ctDNA and these have been extensively reviewed elsewhere1,21. Assay choice is influenced by the sample type1,21. Another key consideration is the relative amount of ctDNA present within a sample, which dictates the required assay sensitivity. ctDNA assays include those designed to detect specific preselected mutations using digital PCR (dPCR)-based techniques, targeted next-generation sequencing (NGS) panels (comprising up to 1,000 genes), or even broader approaches involving whole-exome sequencing or the analysis of global methylation status22. Considerations regarding the choice of assay apply equally to ctDNA derived from plasma and from non-blood sources. However, the high ctDNA to cfDNA ratio within most non-blood samples relative to plasma suggests that these samples might be less susceptible to dilution by non-tumour-derived DNA. Obtaining larger sample volumes, enabling both a greater quantity of ctDNA to be extracted and analysed and an improved level of sensitivity, is also easier when sampling certain fluids such as urine23.

A plethora of alternative sources of ctDNA are available, and several roles for these samples are now emerging (Fig. 1 and Box 1). Each has advantages and disadvantages when compared to ctDNA from blood plasma (Box 3). In the following sections, we discuss existing evidence supporting the utility of ctDNA obtained from non-blood sources and highlight how the information provided from these samples might augment blood-based analysis. Of note, we use the term ctDNA to refer more broadly to these non-blood sources of tumour-derived cfDNA.

Fig. 1: Examples of different cancers that can be investigated using non-blood sources of ctDNA.
figure 1

Owing to the advantages of direct contact with one or more tumour types, non-blood sources of circulating tumour DNA (ctDNA) have certain advantages that could either supplant or complement plasma ctDNA. CNS, central nervous system; NSCLC, non-small-cell lung cancer.

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Urinary ctDNA

Transrenal tumour DNA

The presence of DNA within urine and its potential clinical use have been recognized since the early 2000s. Urine provides a promising source of ctDNA that can be sampled entirely non-invasively9,24. ctDNA within the urine is comprised of two distinct fractions9. The first is transrenal tumour DNA (trtDNA), which originates from plasma and enters the urine through glomerular filtration and is therefore limited in size (typically <250 bp)25. The low concentration of trtDNA has previously restricted the analysis of this type of ctDNA, although these issues have been partially overcome using modern analysis approaches such as NGS23. The second fraction originates from tumour cells shedding directly from the urinary tract and, by virtue of not undergoing renal filtration, can be of larger molecular weight26.

The most notable advantage of analysing urinary ctDNA is the ability to obtain samples entirely non-invasively, without the need for venesection or for a health-care professional to be present during sampling27. This advantage makes serial sampling much simpler and enables samples to be obtained within the patient’s home, which is of particular interest given the focus on ‘virtual’ care following the COVID-19 pandemic28. Urine samples are potentially also less susceptible to dilution of ctDNA with cfDNA from leukocyte lysis29 although cfDNA shed from the urinary tract epithelia might have a similar effect30. Nonetheless, urine has the unique benefit of enabling the direct assessment of DNA released from urological cancers.

Despite these theoretical advantages, urinary ctDNA assays are substantially less developed than their plasma-based alternatives and certain disadvantages exist and should be highlighted. Firstly, trtDNA must pass through the glomerulus, which limits the molecular weight of the fragments to <250 bp. Secondly, the glomerular filtration rate controls the rate of urinary trtDNA accumulation and can be highly variable, particularly in patients receiving systemic anticancer therapy. Indeed, an analysis of urine samples obtained at various times of day suggests substantially lower trtDNA yields from samples obtained <1.5 h after a previous void27. The use of preservatives and optimized storage and transport temperatures is also crucial given the short half-life of unpreserved DNA27,31. A further potential drawback is the often large volume of voided urine and the dilutional effects this will have on ctDNA. Processing of large-volume samples using cfDNA isolation protocols is more technically challenging and, while some studies describe the detection of ctDNA in small volumes of urine (entry volumes of 1–2 ml), others used larger volumes and added a sample concentration step to the workflow30,32,33.

Most research into clinical uses of trtDNA is from patients with non-small-cell lung cancer (NSCLC), testing for alterations in EGFR, including the T790M mutation, and KRAS23,34,35. EGFR mutation status was examined through dPCR and NGS analysis of urine samples from patients receiving third-generation EGFR inhibitors in clinical trials23. In a cohort of 63 patients from the Tiger-X trial23, comparisons with tissue analysis demonstrated that trtDNA had a detection sensitivity of 67–75% depending on the specific mutation subtype, which increased to 80–93% when larger volumes of urine were available (compared with 87–100% for plasma samples)23. Combined analysis of urinary and plasma ctDNA identified 89% of all T790M mutations compared with 75% following tissue-only analyses23. These data highlight a potential synergistic effect of combining different liquid biopsy methods and provide early evidence of concordance between trtDNA and tissue EGFR status. This approach also enables the identification of EGFR alterations that were not detected in tumour samples and could be used to monitor both treatment response and the development of acquired resistance. Similarly, analysis of the presence of KRAS mutations in trtDNA has shown high levels of concordance of around 70–77% relative to tissue sampling, which correlates favourably with plasma ctDNA36, as well as providing prognostic information for patients with elevated KRAS-mutant trtDNA at baseline35,94,95. Analysis of CSF ctDNA has also been demonstrated to provide insights into mechanisms of acquired resistance that might be confined to the CNS and might better represent the genomic landscape of leptomeningeal disease20,96,97. A number of case reports describing patients with melanoma suggest that CSF ctDNA might provide a useful method of monitoring intracranial disease response or identifying CNS-restricted targetable alterations, although these findings have not been validated in larger cohorts thus far89,98,99.

Certain practical elements require further consideration in order for CSF ctDNA-based assays to be used more widely. Lumbar punctures are routinely performed in certain settings, such as in patients with leptomeningeal disease and in those with haematological cancers with CNS involvement, although they are not usually mandated for those with secondary brain parenchymal metastases. Owing to the invasive nature of a lumbar puncture, obtaining samples for translational research (in the absence of a therapeutic indication such as hydrocephalus) might be ethically challenging. Other limitations to the development of ctDNA-based assays for the analysis of CSF include the small sample sizes, biases created by the exclusion of patients for whom a lumbar puncture is contraindicated and technical issues such as contamination with blood during the procedure and variability in sample processing times.

Pleural and peritoneal fluid ctDNA

Pleural and peritoneal fluids provide a thin lubricating layer that reduces friction between pleural linings, abdominal organs and the peritoneum. They are ultrafiltrates of the blood that are relatively enriched in non-haematopoietic cfDNA owing to an absence of peripheral blood cells. Malignant pleural effusions (MPEs) and ascites involve tumour cells that infiltrate the pleura and/or peritoneum via direct, haematogenous or lymphatic spread. The fluid is therefore in close proximity to tumour tissue and might contain ctDNA from tumour cells100.

Even when cytology is negative, MPEs can have a high cfDNA content and can be more amenable to genomic analysis than both tissue and plasma samples18,101. The most sensitive platforms for plasma ctDNA profiling have only 70–80% sensitivity for samples obtained from patients with advanced-stage NSCLC and <50% for those with early stage disease102. Negative results therefore necessitate tissue biopsy confirmation, which can be challenging in those with early stage disease who have a lower tumour burden and possibly poorly accessible primary lesions. An advantage of analysing pleural ctDNA is that samples (obtained via pleural effusion) are immediately available for genetic testing, whereas tissue samples usually require conventional histopathological processing as per standard-of-care diagnostic workflows. Parallel histological analysis of tissue samples and molecular profiling of targetable mutations in ctDNA from pleural effusions might enable earlier detection of targetable alterations and thus permit earlier selection of the most appropriate therapy103. One study used an NGS-based panel of 416 genes to analyse pleural fluid, plasma, and tissue samples and reported higher VAFs and mutation detection rates in pleural fluid cfDNA (98.4% versus 87% in plasma ctDNA)18. Furthermore, 93% of tissue-determined driver alterations were detectable in pleural fluid ctDNA compared with 62% in plasma18. In another study, actionable variants were identified in 87% of MPE samples versus only 48% of paired plasma samples103.

A number of studies have investigated the utility of ctDNA in peritoneal fluid, either obtained from ascites that have developed as a consequence of malignancy or from peritoneal lavage during surgical resection104,105. This approach might be particularly useful for the detection and monitoring of peritoneal disease106,107,108. A study involving 20 patients with CRC and isolated peritoneal disease demonstrated that peritoneal ctDNA was detectable in all patients, whereas only 20% of patients had detectable plasma ctDNA, with significantly higher VAFs in peritoneal cfDNA (16.4% versus 0.28%; P = 0.0019)108. Smaller-cohort studies have investigated ctDNA obtained from presurgical or postsurgical peritoneal lavage as a predictor of disease recurrence, with promising initial results reported107,109.

Similar considerations regarding the feasibility of sample collection apply to both pleural or peritoneal aspiration and CSF sampling: pleural and/or peritoneal aspiration is a moderately invasive procedure requiring a trained operator. Aspiration for diagnostic or therapeutic purposes at initial presentation is the standard-of-care approach in patients with unexplained effusions/ascites and might also be appropriate if effusion occurs on disease relapse or progression; however pre-planned repeat sampling to monitor treatment response might be less practical.

Salivary ctDNA

Saliva is another body fluid that can be non-invasively obtained without requiring the presence of a health-care professional110. Saliva contains cells, proteins and nucleic acids, but is considerably less complex than blood111. The ability to detect germline DNA in saliva has been used in forensic science for decades as well as for certain medical indications: for example, germline BRCA1/2 mutations can be identified in saliva112. Nonetheless, the lower concentrations of ctDNA relative to germline DNA require more advanced detection technologies113. Similar to other non-blood forms of ctDNA, salivary ctDNA (sctDNA) is ideally placed (owing to direct tumour contact via cellular exfoliation or apoptosis) to inform on the status of local tumours such as head and neck cancers. Furthermore, sctDNA can also originate from more distal malignancies either through passive diffusion, active transport or ultrafiltration of ctDNA from the blood across the mucosal membranes114.

Analysis of sctDNA has the ability to differentiate patients with head and neck squamous cell carcinomas (HNSCCs) from those without this disease5. HNSCC is commonly associated with human papillomavirus (HPV) infection. In a PCR-based analysis of plasma and saliva samples from patients with oropharyngeal squamous cell carcinomas, HPV16 DNA was detected prior to treatment with a sensitivity of 52.8% in saliva (67.2% in plasma) and a specificity of 100%115. Furthermore, detectable HPV DNA and the concentration of HPV DNA in saliva following treatment were both associated with a significantly higher risk of disease recurrence (HR 10.7, 95% CI 2.36–48.50; P = 0.002) and inferior OS (HR for death 25.9, 95% CI 3.23–208; P = 0.002)115 and, in a separate study, were demonstrated to correlate with tumour burden and to be predictive of treatment response116. Results from pretreatment and post-treatment samples suggest saliva alone is the ideal sample for the assessment of malignancies of the oral cavity, although this should be used in combination with plasma ctDNA for assessments of tumours located in the oropharynx, hypopharynx and larynx, all of which have less direct exposure to saliva117.

The analysis of sctDNA is limited by several technical challenges118. In patients with NSCLC, dPCR-based analysis of EGFR mutations in plasma and saliva samples revealed high levels of concordance, although EGFR alterations were not detected in saliva DNA samples from several patients with detectable EGFR alterations in plasma. This observation might be explained by the low concentrations of ctDNA entering the saliva from plasma118. Other research suggests that sctDNA fragments are ultrashort (40–60 bp) and might not be amplifiable using conventional PCR techniques; this observation led to the development of an electric field-induced release and measurement (EFIRM) assay to detect EGFR alterations in saliva119,120,121. Direct comparisons suggest that EFIRM is a more sensitive method of detection than dPCR, and might therefore be the optimal method of assessing saliva samples from patients with malignancies other than HNSCC122.

Stool ctDNA

Human DNA is hypothesized to enter the stool via a combination of cellular shedding and colonocyte apoptosis123. Nonetheless, owing to the large and often diverse gastrointestinal microbiome, human DNA accounts for only around 0.01% of the total DNA content of stool, with the remainder derived from bacteria present in the gastrointestinal tract and/or diet123,124.

Screening for CRC is an important current focus of research into the role of stool DNA. This aspect is one of the most advanced areas where non-blood ctDNA testing is being translated into clinical use. The rationale is that early stage colorectal lesions develop predominantly within the mucosa with epithelial shedding of DNA into the lumen of the colon. Analyses of stool DNA samples using sequencing panels comprising 3 mutations in KRAS, 8 in TP53, 10 in APC, BAT-26 and a marker of elongated DNA strands have demonstrated increased sensitivity for detection of CRC compared to faecal immunochemistry and occult blood testing, albeit with an increased risk of false-positive results7,125. This assay has subsequently been developed into a standalone FDA-approved screening test (Cologuard), with evidence of increased uptake among individuals who were previously not compliant with stool screening tests126,127. However, this technique is less cost-effective than the alternatives and therefore might not be appropriate for use in large-scale screening programmes128. The Cologuard assay is also being studied as an alternative to colonoscopic surveillance post removal of polyps/adenomas, which could reduce both costs and burden on patients129.

The utility of stool DNA analysis has also been investigated in patients with other tumour types. Pancreatic cancer is notorious for its usually late diagnosis and poor prognosis; therefore, substantial efforts are currently focused on early diagnosis. Pancreatic secretions directly enter the gastrointestinal tract and the feasibility of detecting KRAS mutations in stool samples obtained from patients with pancreatic adenocarcinomas or cholangiocarcinomas has been studied; five of the six mutations found in stool were identical to those present in the resected carcinomas130. Mutations in KRAS and DNA methylation markers were detected in stool samples from patients with pancreatic cancer using dPCR, although only 19% of patients enrolled in this study agreed to provide a stool sample, highlighting compliance as a limitation131. Indeed, several studies have identified participant aversion to stool-based sampling, which might hinder the adoption of such methods132,133.

Outside of screening, NGS-based analysis of stool DNA samples has been successful in the detection of a range of hotspot mutations in established cancer-associated genes in samples from patients with gastrointestinal cancers, thus highlighting a role for stool DNA analysis in precision medicine and prognostication134. Analysis of stool DNA might also have utility beyond the detection of tumour DNA: analysis of 16S ribosomal DNA in stool samples from patients with NSCLC suggests that the composition of the microbiome can be associated with both responsiveness and toxicities in patients with immune-checkpoint inhibitors and could act as a predictive biomarker135.

In summary, stool DNA has the potential to improve outcomes via analysis as part of cancer screening programmes and to provide information on the genomic profiles of individual tumours, thus informing precision medicine approaches. Current challenges include the rarity of detectable tumour DNA within stool, an insufficient understanding of the stool microbiome and patient aversion to providing faecal samples.

Seminal fluid ctDNA

Seminal fluid, originating from the seminal vesicles and contributing to the liquid component of the male ejaculate, is another bodily fluid that can be used to obtain ctDNA for analysis. Cell-free seminal fluid DNA (cfsDNA) generally exists at higher concentrations relative to cfDNA in other bodily fluids; this relative abundance of cfsDNA might be explained by a combination of sexual abstinence (3–5 days) before providing a sample (permitting secreted DNA to accumulate) and reduced DNase activity owing to the effects of other semen content151. Similarly, in patients with CRC, plasma ctDNA can be detected at substantially lower levels in those with isolated peritoneal disease than in those with liver metastases (in contrast to peritoneal cfDNA)108. Thus, identifying sources of genetic material beyond plasma or tissue might help circumvent these effects of anatomical variations, although crucially, the disease distribution in a particular patient will need to be considered carefully when deciding which assays are likely to yield the most useful results and how best to interpret those results.

The majority of published data available at this point have used mutation-based approaches to study ctDNA and this is reflected in the scope of our Review. The most obvious direct clinical application of these methods currently seems to be the detection of targetable driver or resistance mutations. However, growing research interest exists in the detection of DNA methylation and fragmentation in ctDNA; evidence of the ability to apply such methods to the various biofluids discussed in this Review is still emerging58,62,148,152.

Conclusions

In summary, the analysis of ctDNA obtained from non-blood bodily fluids has several potential benefits, from enabling exploratory research through to routine clinical use, and provides unique benefits such as enabling more detailed analysis of specific tumour types in certain anatomical locations and the potential for entirely non-invasive serial sampling. However, well-designed clinical trials in which the results are used to inform treatment decisions and that demonstrate meaningful benefits to patients will be necessary before non-blood ctDNA assays are to be broadly implemented clinically.