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MOLECULAR BIOMARKERS IN UROLOGIC ONCOLOGY: ICUD-WUOF CONSULTATION

Circulating Tumour DNA as a Biomarker  
Source in Metastatic Prostate Cancer
Gillian Vandekerkhove, Alexander W. Wyatt

Vancouver Prostate Centre, Department of Urologic Sciences, University of British Columbia, Vancouver, Canada

Abstract

Tumour molecular features are increasingly linked to treatment response and patient prognosis in advanced prostate 
cancer. Plasma cell-free circulating tumour DNA (ctDNA) isolated from a minimally invasive blood draw offers 
a convenient source of tumour material to develop clinical biomarkers. Importantly, the burden of ctDNA in the 
blood has strong prognostic implications at different points during the natural history of metastatic progression. 
In prostate cancer, the identification of somatic profiles from ctDNA requires a broad next-generation sequencing 
approach because of the low mutation rate and frequent structural rearrangements. Nevertheless, comparison 
of genomic profiles between liquid and tissue biopsies has demonstrated that ctDNA is a surrogate for tumour 
tissue in the metastatic setting. Our understanding of resistance to androgen receptor (AR) directed therapies 
has been significantly augmented by the frequent detection of AR gene amplifications, mutations, and structural 
rearrangements via liquid biopsy. Furthermore, early studies suggest that distinct molecular subtypes derived 
from ctDNA profiling can help determine the optimal therapeutic regimen for an individual patient and enable  
real-time monitoring for therapy response and resistance. Indeed, in clinical trials targeting the DNA damage repair 
pathway in prostate cancer, ctDNA-based assessment of DNA repair status is already under evaluation as a predictive 
biomarker. Recent advances in the study of circulating DNA fragments now make it possible to interrogate aspects 
of the epigenome. In this review, we describe the various applications of plasma ctDNA in metastatic prostate cancer, 
including its potential role as a clinically informative liquid biomarker.

Etiology of Cell-Free DNA
Upon cell death, genomic DNA fragments can diffuse into sur-rounding bodily fluids. The most familiar source of cell-
free DNA (cfDNA) is peripheral blood, but it can also be purified from urine, sputum, cerebrospinal fluid, and ascites. 
In healthy individuals, the cfDNA in each fluid is derived from cell types in the immediate neighbourhood. Therefore, 
in blood plasma, most cfDNA originates from hematopoietic cells [1,2]. With conventional extraction methodology, 
blood from healthy individuals yields about five nanograms of cf DNA (approximately 750 diploid genomes) per 
milliliter of double-spun plasma [3]. Plasma cfDNA has a periodic 167 base pair fragment pattern consistent with 
apoptotic processing, representing the intervals at which caspase-activated DNase cleaves DNA [4]. The presence of 
high molecular weight DNA in processed plasma is indicative of pre-analytic failure; proper collection, storage, and 
processing of plasma cfDNA is crucial to downstream success [5].

Injury and disease can alter the etiology of cf DNA [1,6,7]. In cancer patients, genomes from tumour cells 
undergoing apoptosis can be shed into body fluids. These tumour-derived cfDNA fragments are termed circulating 
tumour DNA (ctDNA), and they can be detected against a backdrop of cfDNA from benign cells using assays to 
identify somatic alterations or epigenetic marks. The half-life of cfDNA is typically measured in hours but varies by 
the enzymatic activity in each body fluid [8]. Moreover, the kidneys, liver, and spleen all clear cfDNA fragments in 
circulating blood. This rapid turnover means that detection of ctDNA in blood represents a real-time cancer biopsy.

Key Words Competing Interests Article Information

Circulating tumour DNA, cell-free nucleic 
acids, prostatic neoplasms, castration-
resistant, liquid biopsy, recombinational  
DNA repair, DNA mismatch repair,  
androgen antagonists, biomarkers

Alexander Wyatt reports personal fees  
from AstraZeneca, grants and personal  
fees from Jannsen, personal fees from  
Bayer, outside the submitted work.  
Gillian Vandekerkhove reports no  
competing interests.

Received on June 25, 2020 
Accepted on September 8, 2020

Soc Int Urol J. 2020;1(1):39–48 

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40 SIUJ  •  Volume 1, Number 1  •  October 2020 SIUJ.ORG

MOLECULAR BIOMARKERS IN UROLOGIC ONCOLOGY: ICUD-WUOF CONSULTATION

Abbreviations
AR -GSRs androgen receptor genomic structural rearrangements
cfDNA cell-free DNA
ctDNA circulating tumour DNA
HRR homologous recombination repair 
mCRPC metastatic castration-resistant prostate cancer
MMRd mismatch repair deficiency
PARP poly (ADP-ribose) polymerase 
PCa prostate cancer
WGS whole-genome sequencing

Approaches for Cell-Free Circulating 
Tumour DNA Analysis
In cancer patients, the proportion of cf DNA that is 
tumour-derived (ie, the ctDNA fraction) is highly 
variable [9]. This variability cannot be easily estimated 
without profiling purified cf DNA, and represents the 
major technical challenge for ctDNA detection and 
characterization. Assays must be highly sensitive to 
detect the possibility of rare ctDNA fragments diluted in 
hundreds to thousands of normal cfDNA fragments [10].

Polymerase chain reaction-based approaches to detect 
or characterize ctDNA rely on either prior knowledge of 
tumour genotype (eg, from whole-exome sequencing of 
the matched primary tumour) or the plausible presence 
of recurrent hotspot mutations. Unfortunately, with the 
possible exceptions of the AR and SPOP genes, prostate 
cancer (PCa) does not harbour highly recurrent hotspot 
mutations with obvious clinical relevance [11,12], thus 
broader approaches are required to capture the frequent 
genomic structural rearrangements and copy number 
changes [13].

At present, most clinical research using ctDNA in 
PCa has applied targeted next-generation sequencing 
approaches that capture a limited number of exons for 
a set of cancer-related genes [14]. Because of the lower 
cost, targeted sequencing is preferable to conventional  
whole-exome or genome approaches. For tumour tissue-
based analysis, sequencing coverage of 30x to 100x is 
adequate to characterize the somatic genome [15,16]. 
However, ideal sequencing depths for ctDNA are 
typically above 1000x and often considerably higher [10]. 
This can be expensive, and hence the selection of genes 
or regions for cf DNA sequencing is a delicate balance 
between cost, genome coverage, and desired detection 
sensitivity. Most commercial assays cover several 
ubiquitous cancer genes that are relevant for PCa, such 
as TP53, MYC, and BRCA2. However, important PCa 
genes that are not always present in historical pan-cancer 
approaches include SPOP, FOXA1, and CDK12 [13]. 
The selection of regions to sequence within each gene 

must also be carefully considered; inclusion of intronic 
and untranslated regions can enable identification of 
structural rearrangements and gene deletions. In PCa, 
PTEN, RB1, MSH2, FOXA1, and AR are often disrupted 
by structural rearrangements affecting introns [15,17–
20]. Some of these genes can also be perturbed by 
partial or entire locus deletions. From an assay design 
perspective, comprehensive detection of structural 
rearrangement breakpoints requires probes tiled across 
introns. Inclusion of introns (often spanning kilobases) 
in targeted sequencing assays can significantly increase 
the cost of ctDNA profiling.

The ctDNA fraction of a sample determines the type 
of somatic alterations that can be detected. Advances 
in library preparation techniques (eg, duplex unique 
molecular identifiers) and bioinformatic approaches 
(eg, digital error suppression) enable somatic mutation 
identification at ~0.1% frequency [21,22]. However, 
mat hematica l limits regarding t he detection of 
copy number changes cannot be easily overcome by 
technological improvements. For example, the detection 
of entire chromosome arm deletions typically requires a 
ctDNA fraction of at least 5% (rare outside of progressing 
metastatic disease) when using reasonably cost-effective 
targeted sequencing approaches [23]. CtDNA purity must 
be even higher to enable detection of focal deletions. 
In PCa, several focal copy number changes have clear 
clinical relevance (eg, deletions affecting PTEN, MSH2, 
BRCA2) [24]. Therefore, cf DNA assays should report 
ctDNA fraction and discriminate between a true negative 
result (ie, tumour wild type status) and the inability to 
detect a change due to low tumour DNA purity.

I n re s e a rch s e t t i ngs , PC a c f DNA s a mple s 
have been subjected to whole-exome or whole-
genome sequenci ng [18, 23, 25, 26]. W hole-exome 
sequencing is genera l ly cost-justif iable only in 
samples with ctDNA fractions above 20% to 40%, 
but in such scenarios can provide a snapshot of 
somat ic mutat ions a nd copy nu mber cha nges. 
Deep whole-genome sequencing (WGS) of cf DNA 
is not fea sible out side bespoke a na lyses, but  
low-pass WGS is a cost-effective alternative that has 
shown promise for wide uptake [27]. With this method, 
the entire genome is sequenced at a shallow depth, 
normally less than 1x. Low-pass WGS can provide an 
estimate of ctDNA fraction (although typically not 
below 3% to 5%) and yields a low resolution genome-wide 
copy number profile. Since no targeted capture steps are 
required, it is cheap and quick to perform, and software 
packages for data analysis are publicly available [27]. 
However, low-pass WGS does not inform on somatic 
mutations, complex structural rearrangements, or focal 
copy number changes. Furthermore, the continual 
improvement of modular capture assays and targeted 
designs incorporating genome-wide targets means 

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41SIUJ.ORG SIUJ  •  Volume 1, Number 1  •  October 2020

Circulating Tumour DNA as a Biomarker Source in Metastatic Prostate Cancer

that it is possible to incorporate a backbone for whole-
genome copy number profiling (eg, leveraging germline 
single nucleotide polymorphisms) into modern targeted 
sequencing assays.

Idea lly, deep sequencing of plasma cf DNA is 
accompanied by sequencing of matched leukocytes 
as a germline surrogate. In addition to identifying 
pathogenic germline alterations affecting genes such as 
BRCA2 and TP53 [28], leukocyte sequencing also allows 
resolution of somatic mutations in cf DNA related to 
clonal hematopoiesis of indeterminate potential rather 
than cancer [17,29].

In summation, these factors significantly impact 
the detection sensitivity of each cf DNA profiling 
approach, and can lead to divergence in the results when 
comparing commercials tests with one another or with 
research assays [13,30,31]. Mutations with low variant 
allele fractions are particularly unreliable, which is likely 
due to clonal hematopoiesis and somatic expansions. 
End users must be aware of the limits of detection 
for their chosen assay. Ultimately, there is no single 
ctDNA testing approach that can inform on all possible 
scenarios in prostate cancer, and therefore, the choice 
should be governed by the scientific or clinical question 
of the investigator.

Circulating Tumour DNA Abundance as  
a Prognostic Biomarker
T h e  a b u n d a n c e  o f  c t DNA  i s  a  p o t e nt i a l l y 
clinically impactful variable, even without further 
characterization of molecular subtype. In metastatic 
castration-resistant prostate cancer (mCRPC), plasma 
ctDNA abundance is associated with clinical measures 
of disease burden such as prostate-specific antigen level 
and the presence of visceral metastatic lesions [23,26,32–
34]. Accordingly, high ctDNA fractions are associated 
with poor overall survival and short progression-free 
survival in mCRPC patients treated with standard of 
care [23,32,35–37]. The converse is also true: low or 
undetectable ctDNA appears to be a marker of good 
prognosis [23]. Importantly, ctDNA fraction in mCRPC 
appears to provide independent prognostic information 
to standard clinical factors, suggesting that assays of 
ctDNA abundance could become part of prognostic 
models [23,37].

Since ctDNA abundance is closely related to the 
volume of proliferative disease, effective therapy has 
a rapid impact, and therefore blood collection before 
treatment or at clinical progression is recommended to 
maximize the chance of sufficient ctDNA for genomic 
characterization [17,37,38]. In metastatic castration-
sensitive PCa, one week of androgen-deprivation therapy 
can reduce ctDNA fractions by 10-fold [38]. In mCRPC, 

declines in ctDNA are associated with prostate-specific 
antigen responses to abiraterone or enzalutamide, 
and patients with a rising ctDNA fraction while on 
treatment are at greater risk of progression [26,39–41]. 
The detection of changes in ctDNA fraction during 
treatment is a potential surrogate biomarker of response 
and should be explored in prospective biomarker trials.

Relationship of Circulating Tumour DNA  
to Tumour Tissue Biopsy
Tumour molecular features derived from liquid biopsies 
are typically expected to align with those from tissue-
based analyses. In a study of 45 patients with mCRPC, 
deep targeted sequencing of same-day metastatic tissue 
biopsies and plasma cf DNA collections demonstrated 
high concorda nce for t y pica l PCa driver gene 
alterations such as TP53 mutation, AR amplification, 
SPOP mutation, and PTEN deletion [42]. In a parallel 
study, copy number profiles were highly concordant 
when applying low-pass WGS to mCRPC patient-
matched tissue and ctDNA [43]. More recently, high 
tissue-ctDNA concordance for driver gene alterations 
has been reported in de novo metastatic castrate-
sensitive PCa [38], and even among genomically or 
pathologically distinct patient subsets such as those with 
somatic mismatch repair defects or neuroendocrine 
features [18,25]. Collectively, the similarity between 
patient-matched tissue and ctDNA is consistent with the 
findings from rapid autopsy studies in which the vast 
majority of truncal driver alterations were conserved 
across metastatic sites [44,45]. Nevertheless, subclonal 
or late-arising alterations associated with acquired 
treatment resistance (eg, AR amplification or mutation), 
and neutral passenger mutations, are likely to vary 
between metastatic lesions and therefore between a 
single biopsy site and ctDNA.

DNA Damage Repair Defects as Prognostic 
and Predictive Biomarkers
DNA damage repair defects are common in PCa, 
particularly in metastatic disease [46,47], but their 
precise prognostic relevance is contingent on a number 
of factors. For example, there are several distinct DNA 
repair pathways and hundreds of individual genes with 
different degrees of involvement. Alterations in each 
pathway and even gene can have drastically different 
downstream genomic and clinical effects in addition to 
the specific class of alteration observed.

The most commonly affected DNA repair gene in 
mCRPC is BRCA2, which is altered at the germline 
and/or somatic level in ~10% of patients [15,48,49]. 
Biallelic BRCA2 defects result in compromised ability 
to repair double-strand DNA breaks and reliance on 

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alternative repair pathways that are more error-prone 
in this context [50]. Since monoallelic BRCA2 deletion 
is common in PCa, allelic status is key to reporting 
pathogenicity. In mCRPC with high ctDNA fractions, 
loss of heterozygosity across deleterious germline 
BRCA2 mutations is evident in ctDNA, suggesting 
that cf DNA sequencing could identify patients with 
functional BRCA2 loss [51]. It is plausible that broad 
cfDNA sequencing will also be able to identify genomic 
signatures of defective homologous recombination 
repair (HRR), as has been demonstrated by tumour 
tissue sequencing [52,53]. Prospective and retrospective 
studies have suggested that BRCA2 defects detected 
via leukocyte and cf DNA sequencing associate with 
poor mCRPC outcomes in the context of AR-targeted 
therapy [23,37,51,54]. An association between plasma 
ctDNA DNA repair defects and poor outcomes 
has also been obser ved in metastatic castration-
sensitive disease [37]. However, these associations 
appear variable across patient cohorts and were not 
confirmed in some retrospective studies using tumour 
tissue [55,56]. Regard less, mCR PC wit h bia llelic 
BRCA2 defects are vulnerable to therapies exploiting 
defective HRR, such as platinum chemotherapy or poly 
(ADP-ribose) polymerase (PARP) inhibitors [57–62].  
Since a large minority of mCRPC have low levels of 
ctDNA, practitioners must understand the context of a 
‘negative’ result when using liquid biopsies to screen for 
HRR defects. If there is no evidence for ctDNA in the 
sample, then the tumour may still carry somatic HRR 
alterations, and tissue testing should be pursued. Most 
DNA repair defects appear to be truncal to the metastatic 
lineage, so reflex testing of either archival primary tissue 
or metastatic biopsy is appropriate [18,63].

In PCa, ot her HR R genes such as BRCA1, 
PALB2, and RAD51 are altered at frequencies below 
1% [24,48,64]. It is plausible that affected mCRPC 
tumours are vulnerable to PARP inhibitors, but to 
date no clinical trials have been powered to address 
this question. Conversely, ATM and CDK12 mutations 
are prevalent in mCRPC, but their direct association 
with HRR is tenuous, with unique implications for 
PARP inhibitor response. CDK12 mutations are linked 
to a distinct tandem duplicator phenotype and poor 
prognosis with standard of care treatments [65–69]. In  
CDK12-mutant mCRPC, PARP inhibitor response rates 
have been low, regardless of patient selection via liquid 
or tissue biopsy. The frequent tandem duplications in  
CDK12-mutant tumours may result in an elevated 
neoa nt igen burden a nd sensit iv it y to i mmu ne 
checkpoint blockade, but this hypothesis is untested 
in clinical trials [67,68]. Conversely, ATM mutations 
have not been linked to a genomic phenotype. The 
prognosis of mCRPC with ATM mutations is unclear, 
but as with CDK12, response rates to PARP inhibitors 

appear to be reduced in comparison to BRCA2 [62]. 
Currently, prospective plasma ctDNA sequencing is 
under evaluation in several phase II/III PARP inhibitor 
clinical trials in mCRPC, and upon regulatory approval 
is likely to be key for patient biomarker screening. The 
largest hurdle to be overcome for reliance on plasma 
ctDNA screening is the detection of BRCA2 biallelic 
deletions, which (unlike ATM and CDK12) are recurrent 
in mCRPC.

DNA mismatch repair defects are present in 3% to 
5% of mCRPC [18,24]. MSH2 and MSH6 alterations 
predominate and can take the form of complex 
structural rearrangements, thus complicating detection 
strategies [70]. Like HRR-deficient tumours, those with 
mismatch repair deficiency (MMRd) display distinctive 
mutationa l signatures, including hy permutation  
(C > T transitions, particularly in the NCG trinucleotide 
context) and microsatellite instability. MMRd signatures 
can be detected in plasma ctDNA from patients with 
mCRPC [17,18,71]. Although high tumour mutational 
burden is not exclusive to MMRd etiology, in PCa there 
are no other common causes of hypermutation, and 
assays assessing mutational burden in ctDNA can be 
used [18]. Patients with MMRd mCRPC may respond to 
immune checkpoint inhibitors [72].

In the context of DNA repair defects and PARP 
inhibitors, serial sampling can enable resistance 
me c h a n i s m  id e nt i f ic at ion .  BRC A 2  re ve r s ion 
mutations can be detected in plasma ctDNA at clinical 
progression on platinum chemotherapy or PARP 
inhibitors [13,40,73–77]. Plasma cf DNA sequencing 
identifies a greater diversity of BRCA2 reversion 
mutations than biopsy of a single metastatic site [73]. It 
is plausible that regular plasma cfDNA screening could 
detect emerging BRCA2 reversion mutations prior to 
clinical progression, offering opportunities for earlier 
interventions.

AR Mutations, Amplifications, and 
Genomic Structural Rearrangements
Missense mutations in the AR ligand-binding domain 
can alter ligand affinity and drive therapy resistance 
and/or indicate potential vulnerabilities. Overall, AR 
mutations are found in ~10% of ctDNA-positive mCRPC, 
but few point mutations are widely recurrent, principally 
L702H, W742L/C, H875Y and T878A [23,32,33,39,78]. 
AR W742L/C mutations are a resistance mechanism 
to bicalutamide and are frequently identified in the 
plasma ctDNA of bicalutamide treated patients. Next-
generation AR-targeted therapies have activity against 
AR W742L/C, meaning that its detection via liquid 
biopsy may predict durable responses to enzalutamide 
and abiraterone [23,39]. AR T878A and L702H tend to 
arise after therapy, permitting agonism of the AR by 

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progesterones and glucocorticoids, respectively [32,33]. 
While detection of these alterations in plasma cf DNA 
is linked to poor outcomes, switching to different AR-
targeted therapies or steroid regimens may be effective 
in some scenarios [79].

AR copy number gain is the most frequent category 
of AR gene alteration in mCRPC, enabling tumours 
to adapt to low androgen levels during treatment [15]. 
AR copy gain in plasma cf DNA has been associated 
with shorter progression-free survival and overall 
survival in mCRPC patients treated with AR-targeted 
therapy [23,32,37,39,80–84]. However, measuring AR 
copy gain in plasma cfDNA is complicated by variability 
in ctDNA abundance between mCR PC patients. 
Resolving a single extra AR gene copy requires a ctDNA 
fraction of approximately 20%, whereas the signal 
from 8 AR copies can be detected at a ctDNA fraction 
of only 5% [23]. In reality, AR gain is not binary, but 
rather a continuous variable capturing increasing AR 
copies, and an AR copy dose-effect relationship with 
patient prognosis is plausible in the advanced-disease 
setting [23]. Interestingly, plasma AR copy gain ris not 
associated with poor outcomes in mCRPC patients 
treated with taxane chemotherapy, suggesting an 
opportunity for a predictive biomarker [85].

AR copy gain acquisition requires a series of structural 
rearrangements affecting the AR locus. Genomic 
breakpoints falling within the AR itself are termed 
AR-GSRs (genomic structural rearrangements). Some 
AR-GSRs result in a transcript coding for a truncated 
ligand-binding domain, similar in concept to the 
splice variant ARv7 but usually distinct in nucleic acid 
sequence [86,87]. While the downstream consequences 
are challenging to predict from DNA breakpoints alone, 
in vitro studies have suggested that select AR-GSRs 
give rise to constitutively active AR proteins and drive 
therapy-resistant phenotypes. AR-GSRs can be detected 
via ctDNA sequencing of AR introns, and are linked 
to primary resistance to AR-targeted therapies [23,88].  
The presence of AR-GSRs is positively correlated with 
AR copy number [86,89], and AR-GSRs appear to be 
more abundant in patients with late-stage disease than 
initial mCRPC progression [17].

Other Common Genomic Alterations as 
Potential Biomarkers
The tumour suppressor TP53 is altered in over 50% of 
mCRPC [12,48]. TP53 alterations detected in plasma 
cf DNA are linked to worse overall survival and poor 
response to AR-targeted therapy [23,79,89], independent 
of ctDNA fraction and clinical prognostic factors [23]. 
PCa lacking the tumour suppressor triumvirate of 
TP53, RB1, and PTEN are generally clinically aggressive 
and primed for lineage plasticity and rapid adaptation 

to therapy-induced bottlenecks [90,91]. Potentially 
aggressive disease variants can be identified at an early 
stage through the detection of TP53, RB1, and PTEN 
alterations in ctDNA [37]. Conversely, SPOP mutations 
appear to be a good prognostic factor when identified in 
either ctDNA or tissue [23,92].

PTEN deletion is the most common PI3K pathway 
alteration in mCRPC [48]. Other alterations affect this 
pathway, including activating missense mutations in 
AKT1 and PIK3CA in 6% of patients [93]. Tumours 
with PI3K alterations may be reliant on PI3K signalling 
for survival and therefore represent a therapeutic 
vulnerability. PTEN deletion appears to be a biomarker 
for selecting patients most likely to respond to PI3K 
pathway inhibition [94]. Consequently, the pan-Akt 
inhibitor ipatasertib is under evaluation in a phase III 
clinical trial in mCRPC patients with PTEN defects 
(NCT03072238). It is unclear whether other PI3K 
signalling pathway alterations will be relevant for 
ipatasertib response if the drug is approved. However, 
a recent ctDNA-based study suggested that patients 
with somatic truncal hotspot mutations in AKT1 or 
PIK3CA are reliant on the pathway and may have strong 
responses to ipatasertib [93].

Non-Genomic Information Available  
in Cell-Free DNA
In addition to genomic alteration status, ctDNA profiling 
can inform on aspects of the epigenome. Nucleosomes 
protect cf DNA from degradation by circulating 
nucleases, and their positioning can be inferred from 
whole-genome mapping of cfDNA fragments. Patterns 
of nucleosome spacing indicate tissue of origin and were 
crucial in demonstrating that plasma cfDNA is largely 
derived from hematopoietic cells [1]. In addition, plasma 
cf DNA fragmentation patterns vary between cancer 
patients and healthy individuals, indicating diagnostic 
potential [2]. Lastly, the non-random fragmentation 
pattern of ctDNA means that even transcription factor 
activity and gene expression can be inferred from whole-
genome cfDNA sequencing [95,96].

Epigenetic marks, such as cytosine methylation, are 
tissue- and cancer-specific features present on cfDNA/
ctDNA fragments [97,98]. Therefore, tissue of origin 
can also be predicted through methylation profiling 
of cfDNA [7,99]. Importantly, the number of cell type-
specific methylation marks in a tumour cell vastly 
outnumbers the somatic mutation count. Therefore, 
plasma cf DNA methylation assays have potential 
for greater ctDNA detection sensitivity than assays 
reliant on capturing somatic mutations, especially in 
the context of early cancer diagnosis [97,98]. Detection 
of prostate lineage methylation marks on cf DNA can 
provide an accurate measure of ctDNA fraction and 

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even resolve patients with AR copy gain [100]. Finally, a 
recent study demonstrated that in a subset of mCRPC 
patients with high ctDNA fractions, simultaneous 
whole-exome sequencing and whole-genome bisulfite 
sequencing can identify neuroendocrine PCa [25].

Future Directions
True clinical translation of recent ctDNA correlative 
research will require prospective clinical trials. In the 
context of DNA repair and PARP inhibition, cf DNA 
analysis is part of patient screening protocols in several 
ongoing phase II/III trials [61]. The innovative ProBio 
clinical trial (NCT03903835) is an outcome-adaptive, 
multi-arm, platform trial testing the utility of liquid 
biopsies to tailor treatment decisions in mCRPC [101]. 
The initial arms in this trial will test prognostic and 
predictive biomarkers for many of the current standard 
of care therapies such as abiraterone and cabazitaxel. 
PC-BETS (NCT03385655; also known as IND234) 
is another multi-arm umbrella trial, but set in a later 
stage than ProBio, testing investigative agents such as 
adavosertib, darolutamide, palbociclib, and ipatasertib. 
CtDNA fraction as a potential biomarker is also being 
prospectively tested in the phase II PROTRACT trial 
(NCT04015622). In PROTR ACT, mCRPC patients 
who have progressed on abiraterone are randomized 

to physician’s choice of enzalutamide or docetaxel, or a 
biomarker-driven stratification based on pre-treatment 
ctDNA fraction. The results from these and similar trials 
will be crucial for moving cf DNA profiling towards 
routine clinical use in mCRPC.

Beyond prospective validation, there are several other 
hurdles that must be overcome before ctDNA analysis 
will become part of standard clinical care. With the 
sensitivity of current technology, liquid biopsy will 
remain uninformative for somatic alterations in those 
patients with minuscule plasma ctDNA levels, meaning 
that tumour tissue testing must remain part of the 
molecular diagnostics paradigm. Teaching practitioners 
how to interpret liquid biopsy results will be key to 
development of workf lows that optimally use both 
ctDNA and tissue testing at appropriate times during 
PCa progression. Furthermore, the current range of 
commercially available ctDNA tests are not tailored for 
PCa, and most of the translational studies described 
in this review leveraged bespoke research-based 
sequencing approaches that cannot be implemented in 
a large clinical system. Therefore, the development of 
clinical-grade liquid biopsy assays that are specific for 
the unique genomic features of PCa will help speed the 
uptake of ctDNA testing in the real world.

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