








































This is an open access article under the terms of a license that permits non-commercial use, provided the original work is properly cited.  
© 2023 The Authors. Société Internationale d'Urologie Journal, published by the Société Internationale d'Urologie, Canada.

SIUJ.ORG SIUJ  •  Volume 4, Number 4  •  July 2023

Key Words Competing Interests Article Information

Testicular cancer, germ cell tumors, cell-free 
DNA, circulating tumor DNA, liquid biopsy

None declared. Received on December 7, 2022 
Accepted on May 13, 2023 
This article has been peer reviewed.

Soc Int Urol J. 2023;4(4):287–292

DOI: 10.48083/IPCO3495

293

REVIEW — LIQUID BIOPSY

The Role of Circulating Tumor DNA and Cell-Free 
DNA in the Management of Germ Cell Tumors:  
A Narrative Review

Isabella Dolendo,1 Shanice Cox,2 Dhruv Puri,1 Aditya Bagrodia1,3

1Department of Urology, UC San Diego School of Medicine, La Jolla, United States 2 Department of Urology, Burnett School of Medicine at Texas Christian University, 
Fort Worth, United States 3 Department of Urology, University of Texas Southwestern Medical Center, Dallas, United States

Abstract

Liquid biopsy has demonstrated success as a diagnostic, prognostic, and therapy response monitoring tool in various 
cancers and could represent a rapid and minimally invasive alternative or complementary test for testicular germ cell 
tumors (GCTs). This article aims to review the current state of the research into circulating tumor DNA (ctDNA) and 
cell-free DNA (cfDNA) in testicular GCTs.

Studies have confirmed the presence of ctDNA and cfDNA can be identified in peripheral blood samples of patients 
with testicular GCTs. Further research has attempted to optimize the methods for ctDNA detection in plasma to 
improve the sensitivity of these tests; however, a single method with high sensitivity and reliability has yet to be 
established. Previous studies have employes different methods for detecting cfDNA, including spectrophotometry, 
capillary electrophoresis, quantitative polymerase chain reaction (PCR), reverse transcription-polymerase chain 
reaction (RT-PCR), and whole genome sequencing. These studies have various elements of cfDNA examined such as 
total cfDNA quantity, methylation patterns, and specific mutations. Additional studies have investigated the efficacy 
of cfDNA detection in combination with other tests including miRNA analysis.

The application of cfDNA as a biomarker has been rapidly expanding in several malignancies. However, there is a 
relative paucity of research on the clinical utility of cfDNA in testicular cancer, and many questions remain about 
the significance and feasibility of this biomarker in GCTs. Cell-free DNA shows promise as a biomarker to enhance 
detection and disease monitoring in testicular cancer, but robust studies are needed to develop an optimal and 
reproducible method for cfDNA detection in order to determine its clinical application in testicular cancer.

Introduction

The diagnostic evaluation of testicular cancer typically involves serum tumor markers including alpha-fetoprotein 
(AFP), human chorionic gonadotropin (hCG), and lactate dehydrogenase (LDH)[1]. However, these markers are 
elevated in only about 40% of patients with testicular cancer[2]. Testicular biopsy is typically avoided because of the 
associated risk for tumor cell seedling and altered patterns of metastases[3]. To overcome these limitations, liquid 
biopsy has emerged as a recent development, providing a rapid, accurate, and noninvasive alternative to tissue biopsy 
and radical orchiectomy[4].

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is considered to be tumor specific and uniquely 
identifiable, and its analysis can provide insights into 
disease burden and progression. Of the various cfDNA 
characteristics, cf DNA methylation and mutation 
analysis hold the most promise as cancer biomarkers[11].

Several studies have identified cf DNA in plasma 
samples of testicular cancer patients. Ellinger et al. 
collected serum samples from patients with semino-
mas or nonseminomas and from healthy patients and 
isolated the cfDNA[1]. The investigators used RT-PCR to 
examine beta-actin DNA fragments (106 bp, 193 bp, and 
384 bp) and found these fragments to be significantly 
increased in patients with testicular germ cell tumors 
(TGCTs) compared to disease-free controls. Boublikova 
et al. investigated total cf DNA using spectrophotome-
try, capillary electrophoresis, and quantitative poly-
merase chain reaction (qPCR) in the peripheral plasma 
of patients with TGCTs and controls[12]. They found 
that patients with TGCTs had significantly higher total 
cfDNA compared to controls.

Studies have shown CTCs can also be detected in the 
peripheral samples of patients with TGCTs. Bokemeyer 
et al. detected CTCs in apheresis samples from 58% of 
patients with metastatic TGCTs undergoing high-dose 
chemotherapy and peripheral blood stem-cell (PBSC) 
transplantation[13]. Fan et al. detected beta-hCG mRNA 
in apheresis products using PCR[14], suggesting the 
presence of a significant number of CTCs or cell-free 
DNA rather than tumor cells. All patients with circulat-
ing mRNA exhibited elevated serum beta- hCG levels, 
whereas only 46% of patients without circulating mRNA 
tested positive for beta- hCG. The findings also revealed 
a correlation between positive PCR results and higher 
serum beta- hCG levels at diagnosis[14]. Similarly, in a 
study by Hautkappe et al., all patients with circulating 
beta- hCG or AFP mRNA had elevated serum beta- hCG 
levels, while only 40% of patients with negative PCR 
results had positive serum beta- hCG[15].

A study conducted by Hildebrandt et al. demon-
strated that reverse transcription-polymerase chain 
reaction (RT-PCR) targeting germ cell alkaline phospha-
tase is highly sensitive in detecting residual GCT cells 
in peripheral blood. This method enabled the detection 
of one tumor cell in at least 106 mononuclear cells[16]. 
Yuasa et al. also used RT-PCR to detect malignant cells 
in the bloodstream by measuring the expression of AFP 
in the peripheral blood of patients with advanced stage 
testicular cancer. They found that this assay could detect 
a single cancer cell in 106 peripheral blood stem cells[17].

Further advancements in research have led to the 
development of techniques that enhance the detectabil-
ity of ctDNA and CTCs in GCTs. Nastały et al. devel-
oped a new assay using a label-free enrichment technique 
based on the physical properties of tumor cells. By using 

TABLE 1. 

List of testicular cancer MeSH entry terms and  
cell-free DNA–related search terms used to identify 
relevant literature in the Cochrane Review and  
PubMed databases 

Testicular cancer MeSH 
entry terms

Cell-free DNA–related search 
terms 

Testicular Neoplasm
Neoplasm, Testicular
Testicular Tumors
Neoplasms, Testis
Neoplasm, Testis
Testis Neoplasm
Testis Neoplasms
Testicular Tumor
Tumor, Testicular
Tumors, Testicular
Neoplasms, Testicular
Cancer of Testis
Testis Cancer
Cancer, Testis
Cancers, Testis
Testis Cancers
Cancer of the Testes
Cancer of the Testis
Testicular Cancer
Cancer, Testicular
Cancers, Testicular
Testicular Cancers
Tumor of Rete Testis
Rete Testis Tumor
Rete Testis Tumors
Testis Tumor, Rete
Testis Tumors, Rete

Biopsies, Liquid
Biopsy, Liquid
Liquid Biopsies
Cell-Free Nucleic Acids
Nucleic Acids, Cell-Free
Circulating Cell-Free Nucleic Acid
Circulating Cell-Free Nucleic Acid
Circulating Nucleic Acids
Acids, Circulating Nucleic
Nucleic Acids, Circulating
Cell-Free Nucleic Acid
Cell-Free Nucleic Acid
Nucleic Acid, Cell-Free
Circulating Cell-Free Nucleic Acids
Circulating Cell-Free Nucleic Acids
Circulating Nucleic Acid
Acid, Circulating Nucleic
Nucleic Acid, Circulating
Cell-Free DNA
Cell-Free DNA
DNA, Cell-Free
cfDNA
cirDNA
Cell-Free Deoxyribonucleic Acid
Acid, Cell-Free Deoxyribonucleic
Cell-Free Deoxyribonucleic Acid
Deoxyribonucleic Acid, Cell-Free
Circulating DNA
DNA, Circulating
Cell-Free RNA
Cell-Free RNA
RNA, Cell-Free
cfRNA
cirRNA
Cell-Free Ribonucleic Acid
Acid, Cell-Free Ribonucleic
Cell-Free Ribonucleic Acid
Ribonucleic Acid, Cell-Free
Circulating RNA
RNA, Circulating
DNA, Circulating Tumor
Tumor DNA, Circulating
Cell-Free Tumor DNA
Cell-Free Tumor DNA
DNA, Cell-Free Tumor
Tumor DNA, Cell-Free

cfDNA: cell-free DNA; cirRNA: circulating tumor DNA;  
MeSH: Medical Subject Heading.

Liquid biopsy involves the extraction and analysis 
of biological samples, such as blood, urine, or saliva. 
Unlike tissue biopsy, which provides information 
confined to a specific region and fails to capture complex 
tumor heterogeneity, liquid biopsy isolates and analyzes 
tumor-derived or tumor-associated components that 
circulate in the bloodstream: circulating tumor cells 
(CTCs), circulating leukocytes, and tumor-derived 
circulating nucleic acids, such as cell-free DNA (cfDNA), 
circulating tumor DNA (ctDNA), microRNA (miRNA), 
and noncoding RNA. This enables longitudinal moni-
toring of cancer progression[4].

Cell-free DNA refers to fragmented DNA in the 
noncellular component of blood. Circulating tumor 
DNA consists of DNA fragments of 150 to 200 base pairs 
(bp) released into the peripheral blood from cancerous 
cells as a result of apoptosis, necrosis, or secretion[5]. The 
presence of tumor-specific genetic and epigenetic alter-
ations in ctDNA makes ctDNA a promising biomarker. 
The concentration of ctDNA in blood plasma varies 
among cancer patients based on the type, location, and 
stage of the cancer and is typically low. The detection of 
somatic mutations, frequently single base-pair changes, 
copy number variations, or chromosomal rearrange-
ments in ctDNA shows promise for early cancer diag-
nosis, tumor dynamics assessment, minimal residual 
disease identification, and therapy monitoring.

Cell-free DNA isolated from cancer patients can be 
used for various downstream applications, such as the 
investigation of mutations, copy number variations, 
gene fusions, and DNA methylation using various meth-
ods including sequencing- or PCR-based approaches. 
ctDNA analysis can potentially overcome tumor hetero-
geneity, which may not be captured by tissue sampling.

Currently, there are 3 FDA-approved liquid biopsy 
tests: 2 cf DNA-based tests (Cobas EGFR Mutation 

Test v2 and Epi proColon) and one CTC-based test 
(CellSearch)[5]. Numerous ongoing clinical trials are 
employing cf DNA analysis in urologic malignan-
cies. Mutations in fibroblast growth factor receptor-3 
(FGFR3) in cf DNA were monitored in patients with 
advanced urothelial carcinoma undergoing treatment 
with an FGFR antagonist[6]. Patients with a decrease 
in mutations in cf DNA were found to correlate with a 
greater decrease in tumor size. Additionally, analysis 
of FGFR3 mutations in cf DNA were shown to predict 
disease progression ahead of imaging with computer-
ized tomography (CT) scans. cfDNA has also been used 
in a study on a programmed death-ligand 1 (PD-L1) 
inhibitor in urothelial cancer by Powles et al.[7]. Those 
authors found a correlation between mutations found in 
ctDNA with mutations found in tumor DNA, suggesting 
a role for plasma-based biomarker screening in predict-
ing immunotherapy response[7].

Cell-free and circulating tumor DNA are currently 
being used in clinical trials for urothelial carcinomas. 
For example, Huang et al. are recruiting for a study 
using ctDNA and urine tumor DNA as biomarkers 
for minimal residual disease and upper urinary tract 
urothelial carcinoma recurrence[8]. Van der Heijden et 
al. are monitoring ctDNA during follow-up of patients 
with urothelial carcinoma treated with ipilimumab and 
nivolumab[9]. Lolkema and van der Veldt are collecting 
ctDNA as part of their study on biomarkers in patients 
with advanced urothelial cancer treated with pembroli-
zumab[10]. In this paper, we review the current state of 
research into liquid biopsies in GCTs.

Methods
A narrative review was conducted on September 24, 
2022, by searching the PubMed and Cochrane Review 
databases without any limitation on the date range. 
Articles addressing cf DNA and ctDNA in testicular 
cancer were included in the review. The search terms 
used included all Medical Subject Heading (MeSH) 
entry terms for testicular cancer and cell-free DNA–
related terms (Table 1). Relevant articles were also 
identified through the references of the included articles. 
All types of studies related to ctDNA and cf DNA in 
testicular cancer were included. Duplicate articles were 
excluded. Three reviewers (I.D., S.C., D.P.) independently 
screened the articles for inclusion. Any discrepancies 
were discussed to determine the final inclusion.

Circulating Tumor DNA and Cell-Free DNA  
in Testicular Cancer
Testicular cf DNA is thought to result from apoptosis 
during spermatogenesis. This can occur in both 
physiological and pathological conditions, including 
ma lignancy. In the case of ma lignancy, cf DNA 

Abbreviations 
AFP alpha-fetoprotein
bp base pairs 
CTCs circulating tumor cells 
cfDNA cell-free DNA 
ctDNA circulating tumor DNA
GCTs germ cell tumors
hCG human chorionic gonadotropin 
LDH lactate dehydrogenase 
miRNA microRNA 
PCR polymerase chain reaction
qPCR quantitative polymerase chain reaction 
RT-PCR reverse transcription-polymerase chain reaction 
TGCTs testicular germ cell tumors 

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REVIEW — LIQUID BIOPSY The Role of Circulating Tumor DNA and Cell-Free DNA in the Management of Germ Cell Tumors: A Narrative Review

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mutations. Tsui et al. identified cf DNA mutations in 
plasma samples and employed whole genome sequenc-
ing to estimate the cfDNA tumor fraction[24]. By using 
cf-IMPACT, a tumor test capable of detecting genomic 
alterations in specific cancer-related genes, the research-
ers found somatic mutations in 60% of samples from 
patients with TGCTs (n = 118). For the remaining 
samples without detected alterations and low tumor 
fraction, a more sensitive but less comprehensive test, 
MSK-ACCESS, was used for re-analysis. This latter 
test showed 14 additional samples with somatic muta-
tions. Whole exome sequencing was then performed 
for samples without detected alterations but high tumor 
fraction, leading to the identification of 5 more samples 
with somatic mutations. In total, 90 of 118 samples 
(79%) exhibited somatic mutations across all 3 meth-
ods. Overall, this study showed that cfDNA tumor frac-
tion can guide further testing for somatic mutations in 
cfDNA to maximize mutation identification. Moreover, 
the identification of specific mutations has the potential 
to inform the selection of precision medicine treatments.

The prognostic implications of the presence of cfDNA 
and ctDNA in GCTs remain unclear. Ellinger et al. 
found that levels of cell-free mtDNA did not correlate 
with disease severity, including tumor stage or lympho-
vascular invasion, and Bokemeyer et al. showed that 
the presence of CTCs in transplanted PBSC apheresis 
products did not affect overall survival[13,23]. On the 
other hand, Hautkappe et al. demonstrated that 41.2% of 
patients with stage IIc/III GCTs had detectable mRNA 
of either AFP or beta- hCG compared to only 23.5% of 
patients with stage I disease[15].

In a clinical trial conducted by Reid et al., cell-free 
DNA is being investigated to determine whether cfDNA 
is detectable in the plasma of patients with platinum 
refractory/resistant GCTs. The researchers are further 
analyzing patients with detectable cf DNA for changes 
in cfDNA that are associated with clinical resistance to 
platinum (NCT0398087)[25].

The clinical management of TGCT has experienced 
limited progress in recent decades in integrating molec-
ular diagnostic and prognostic tools into targeted thera-
pies for affected patients[26]. Total cfDNA and changes 
in cfDNA levels have been shown to be associated with 
specific treatment responses, prognoses, and survival 
rates in various tumors including lung, pancreatic, 
colorectal, and other malignancies.

While the utility of cfDNA in GCTs remains uncer-
tain, circulating miR-371a-3p has recently captured 
attention as a robust biomarker with significant poten-
tial to address clinical gaps in GCT management. In 
2011, Murray and colleagues reported detectable serum 
miR-371a-3p in a 4-year-old boy with disseminated 
yolk sac tumor[27]. Subsequent to this, a large body 

of literature has consistently illustrated the utility of 
miR-371a-3p in differentiating GCTs from controls[28]. 
In the largest report to date, Dieckmann et al. analyzed a 
cohort of 616 GCT patients and found that miR-371a-3p 
exhibited a sensitivity of 90.1% and specificity of 94% 
for the diagnosis of GCT[29]. miR-371a-3p also demon-
strated a sensitivity of 83% and specificity of 96% for 
identifying relapses, indicating its expanded clinical 
utility beyond diagnosis[29]. The predictive capability of 
miR-371a-3p for identifying relapses has been substan-
tiated by Nappi et al. and Lafin et al.[30,31]. Leão et al. 
expanded on previous work by demonstrating that 
miR-371a-3p could also detect residual viable GCT after 
chemotherapy, with an area under the curve of 0.874, 
and with levels declining in response to treatment[32]. 
Apar t from identif y ing teratomas, miR-371a-3p 
shows potential as an important adjunct in the clini-
cal management of GCTs. Currently, 2 clinical trials 
(NCT04914026, NCT04435756) are exploring the role of 
miR-371 in the management of GCTs[33,34]. 

Discussion
The utility of circulating tumor DNA and cell-free DNA 
as diagnostic, prognostic, and therapeutic markers 
has been well-documented in various cancers. While 
the application of cf DNA as a biomarker has been 
rapidly expanding in several malignancies, relatively 
little research exists on the clinical utility of cfDNA in 
testicular cancer, leaving many questions unanswered 
about the significance and feasibility of this biomarker 
in TGCTs.

Studies have demonstrated the presence of ctDNA 
and cfDNA in peripheral blood samples of patients with 
TGCTs. Efforts have been made to optimize the methods 
for ctDNA detection in plasma to enhance the sensitivity 
of these tests; however, a single method with high sensi-
tivity and reliability has yet to be established. Previous 
studies have employed diverse methods for detecting 
cfDNA, including spectrophotometry, capillary electro-
phoresis, qPCR, RT-PCR, and whole genome sequenc-
ing. These studies have also examined various aspects 
of cf DNA, such as total cf DNA quantity, methylation 
patterns, and specific mutations (Table 2). Some stud-
ies have evaluated the efficacy of cf DNA detection in 
combination with other tests including miRNA analysis.

Many questions remain about the implementation 
of cfDNA and ctDNA in the clinical setting. To achieve 
the clinical validity and utility of liquid biopsy tests, 
it is necessary to assess the analytical validity of these 
tests, and then conduct prospective studies using the 
protocols that resulted in analytical validity. Analytical 
validity encompasses the accuracy, sensitivity, specific-
ity, and robustness of the liquid biopsy test and relies on 
pre-analytical variables and protocols used for sample 

keratins 8, 18, and 19, and EpCAM as epithelial cell 
markers and SALL4 andOCT3/4 as germ cell markers, 
they achieved the detection of CTCs in 17.5% of periph-
eral blood samples from testicular cancer patients, 
compared to only an 11.5% detection rate using the 
CellSearch assay[18]. Ruf et al. demonstrated the detec-
tion of CTCs through alkaline phosphatase enzymatic 
activity and the use of anti-keratin, anti-EpCAM, and 
anti-SALL4 antibodies in immunocytochemistry[19].

Taken together, these studies highlight the wide vari-
ety of tests developed to measure cf DNA and ctDNA 
in the peripheral samples of patients with TGCTs. 
However, the inability to designate a single test as the 
gold testing standard hinders comparison of cfDNA and 
ctDNA levels in TGCT patients across different stud-
ies. The variation in testing methods poses a significant 
challenge in establishing clinical application of cfDNA 
and ctDNA in testicular cancer care.

Clinical Utility of Circulating Tumor DNA 
and Cell-Free DNA in Testicular Cancer
Circulating tumor and cell-free DNA offer multiple 
clinical applications in the context of testicular cancer, 
showing promise as diagnostic and prognostic tools. 
Various detection methods have been described in 
the literature. For example, Ellinger et al. used RT-
PCR to isolate beta-actin DNA fragments in cf DNA, 
achieving a sensitivity of 84% and specificity of 97% 
in distinguishing patients with GCTs from healthy 
controls[1]. Even among patients with normal ranges 
of serum tumor markers, cfDNA exhibited a sensitivity 
of 84% and specificity of 97%, suggesting its potential 
utility as a biomarker for GCTs, particularly in those 
patients who do not have elevations in other serum 
tumor markers[1].

Investigations have also focused on total cfDNA and 
its 2 main fragments (360 bp and 180 bp) as targets for 
detection. Boublikova et al. used spectrophotometry, 
capillary electrophoresis, and qPCR to identify total 
cfDNA and found that the quantity of cfDNA does not 
offer a clear threshold to distinguish between TGCT 
patients and controls, thus limiting the sensitivity of 
this marker. However, they found that longer cf DNA 
fragments (360 bp) were present in 58% of patients with 
TGCTs but absent in controls[12], suggesting that target-
ing longer cfDNA fragments instead of total cfDNA may 
be a viable approach. Further studies involving larger 
samples are necessary to establish an acceptable cutoff 
value.

Methylation patterns in serum samples of patients 
with TGCTs have also shown promise as a biomarker. 
Lobo et al. evaluated hypermethylated RASSF1A in 
cfDNA as a diagnostic marker for testicular GCTs[20]. 

RASSF1A is a tumor suppressor gene that exhibits 
inactivation in many cancers. Promoter hypermethyl-
ation of RASSF1A has been a useful marker in several 
solid malignancies and has also been detected in tissue 
samples of TGCTs[20]. To quantify hypermethylated 
RASSF1A, Lobo et al. used a novel droplet digital PCR 
method to analyze 102 serum samples from patients with 
GCTs and 29 samples from healthy young adult men[20]. 
They found that hypermethylated RASSF1A exhibited a 
sensitivity of 86.7%, surpassing the 65.5% sensitivity of 
AFP and hCG markers. They also found that combining 
hypermethylated RASSF1A with miR-371a-3p, a reliable 
microRNA biomarker for TGCTs, exhibited a sensitivity 
and specificity of 100%. This study identified a cfDNA 
target with high sensitivity and specificity, particularly 
in combination with a microRNA marker.

Ellinger et al. also examined hypermethylation 
at RASSF1A, as well as methylation at APC, GSTP1, 
PTGS2, p14(ARF), and p16(INK)[21] using RT-PCR to 
evaluate samples from 73 patients with TGCTs and 35 
individuals without disease. They found significantly 
higher rates of hypermethylation in the patients with 
testicular cancer across all examined gene sites. When 
combining multiple gene sites, the test achieved a sensi-
tivity of 67% and specificity of 97%, surpassing the 
58% sensitivity of conventional biomarkers (AFP, hCG, 
PLAP, LDH). In patients with normal laboratory results, 
the hypermethylation pattern test demonstrated a sensi-
tivity of 97%, indicating its potential as a set of methyl-
ation markers particularly useful for identifying disease 
in patients with normal laboratory markers.

Kawakami et al. identified another potential cfDNA 
biomarker, the 5’ end of the XIST gene expressed in 
male germ cells[22]. This gene has been found to be 
hypomethylated in TGCTs. The investigators analyzed 
plasma samples from 24 patients with TGCTs and 24 
controls and found 64% of patients with TGCTs had 
detectable unmethylated signals compared to none 
of the control samples. The presence of unmethylated 
signals was more common in patients with metastatic 
tumors (88%) compared to patients with nonmetastatic 
tumors (53%). Further research is needed to improve the 
sensitivity of tests for this marker and establish unmet-
hylated XIST fragments as a diagnostic tool for TGCT. 
Ellinger et al. also investigated circulating cell-free mito-
chondrial DNA in the serum of patients with seminoma 
and nonseminomas and found that cell-free mtDNA 
exhibited a sensitivity of 59.6% and 94.3%, respectively, 
in identifying patients with GCTs[23]. For patients 
without elevated serum tumor markers, cell-free mito-
chondrial DNA demonstrated a sensitivity of 64.5% and 
specificity of 91.4%.

Studies have investigated the use of the cell-free 
DNA tumor fraction and the identification of somatic 

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RNA1. Cancer Res.2000;60(12):3170–3174. PMID: 10866307.

18. Hildebrandt MO, Bläser F, Beyer J, Siegert W, Mapara MY, Huhn 
D, et al. Detection of tumor cells in peripheral blood samples from 
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19. Yuasa T, Yoshiki T, Tanaka T, Isono T, Okada Y. Detection of circulating 
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20. Nastał y P, Ruf C, Becker P, Bednarz-Knoll N, Stoupiec M, Kavsur R, et 
al. Circulating tumor cells in patients with testicular germ cell tumors. 
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CCR-13-2819. PMID: 24634372.

21. Ruf C, Nastał y P, Becker P, Isbarn H, Honecker F, Pantel K, et al. 703 
Circulating tumor cells can be detected in patients with testicular germ 
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preparation and biomarker detection[35]. Pre-analytical 
variables are factors not associated with the disease that 
can impact the integrity of the bodily fluids or biomark-
ers present therein and hence inf luence the analysis 
results. These variables can have a technical, biological, 
or environmental origin. The impact of pre-analytical 
variables is based on the biomarker and nature of the 
bodily fluid studied and should be thoroughly assessed 
for each newly developed liquid biopsy test[36,37]. 
Standard operating procedures for the complete work-
flow of liquid biopsy tests are critical for advancing their 
clinical implementation[5].

Despite the success of cf DNA in multiple cancers, 
particularly lung and colon cancers, only 3 liquid 
biopsy tests have received FDA approval. The lack of 

TABLE 2. 

Summary of studies included in this narrative review highlighting the various detection methods and targets

Study Detection method Target

Bokemeyer et al.[13] RT-PCR
beta- hCG, fibronectin (EDB variant), EGFR, CD44 (v8 to 10 variant), germ cell  
and placental alkaline phosphatase, human endogenous retrovirus type K (ENV 
and GAG), and XIST

Boublikova et al.[12]
Spectrophotometry, capillary 
electrophoresis, qPCR

Total cfDNA and its 2 main fragments (360 bp and 180 bp)

Ellinger et al.[23] RT-PCR cell-free mitochondrial DNA

Ellinger et al.[1] RT-PCR 106 bp, 193 bp, and a 384 bp beta-actin DNA fragment

Ellinger et al.[21] RT-PCR
Hypermethylation at RASSF1A; methylation at APC, GSTP1, PTGS2, p14(ARF), 
and p16(INK)

Fan et al.[14] PCR beta- hCG mRNA

Hautkappe et al.[15] RT-PCR beta- hCG mRNA, AFP mRNA

Hildebrandt et al.[16] RT-PCR Germ cell alkaline phosphatase

Kawakami et al.[22] PCR Methylation of 5’ end of the XIST gene

Lobo et al.[20] Droplet digital PCR Hypermethylated RASSF1A

Nastał y et al.[18]
Label-free enrichment technique, 
immunocytochemical staining

Germ cell tumor (anti-SALL4, anti-OCT3/4) and epithelial cell–specific (anti-
keratin, anti-EpCAM) antibodies

Ruf et al.[19] Immunocytochemistry Anti-keratin, anti-EpCAM, and anti-SALL4 antibodies

Tsui et al.[24]
cf-IMPACT, MSK-ACCESS, whole 
genome sequencing

cfDNA tumor fraction and somatic mutations

Yuasa et al.[17] RT-PCR AFP

cfDNA: cell-free DNA; mRNA: messenger RNA; PCR: polymerase chain reaction; RT-PCR: transcription-polymerase chain reaction.

reproducibility in preclinical research may contribute to 
this poor clearance rate by the FDA. Future directions 
may focus on developing standardized methods for 
measuring cf DNA in plasma to ensure reliable results 
that provide clinical value. Standardization efforts may 
also help establish cutoff values for screening and prog-
nostic purposes. Addressing the challenges of cf DNA 
detection, such as the low quantities of cfDNA in plasma 
and its rapid clearance of cfDNA to circulation, is crucial 
for the successful implementation of standardized meth-
ods. Cell-free DNA holds promise as a biomarker to 
enhance detection and disease monitoring in testicu-
lar cancer, but robust studies are needed to establish an 
optimal and reproducible method for cfDNA detection 
before determining its clinical application in the context 
of testicular cancer.

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22. Lobo J, van Zogchel LMJ, Nuru MG, Gillis AJM, van der Schoot CE, 
Tytgat GAM, et al. Combining hypermethylated RASSF1A detection 
using ddPCR with miR-371a-3p testing: an improved panel of liquid 
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(Basel).2021;13(20):5228. doi: 10.3390/cancers13205228. PMID: 
34680375; PMCID: PMC8534014.

23. Ellinger J, Albers P, Perabo FG, Müller SC, von Ruecker A, Bastian 
PJ. CpG island hypermethylation of cell-free circulating serum DNA 
in patients with testicular cancer. J Urol.2009;182(1):324–329. doi: 
10.1016/j.juro.2009.02.106. PMID: 19447423.

24. Kawakami T, Okamoto K, Ogawa O, Okada Y. XIST unmethylated 
DNA fragments in male-derived plasma as a tumour marker for 
testicular cancer. Lancet.2004;363(9402):40–42. doi: 10.1016/S0140-
6736(03)15170-7. PMID: 14723995.

25. Ellinger J, Albers P, Müller SC, von Ruecker A, Bastian PJ. Circulating 
mitochondrial DNA in the serum of patients with testicular germ cell 
cancer as a novel noninvasive diagnostic biomarker. BJU Int.2009 
Jul;104(1):48–52. doi: 10.1111/j.1464-410X.2008.08289.x. PMID: 
19154496.

26. Tsui DWY, Cheng ML, Shady M, Yang JL, Stephens D, Won H, et al. 
Tumor fraction-guided cell-free DNA profiling in metastatic solid tumor 
patients. Genome Med.2021;13:96. doi: 10.1186/s13073-021-00898-8. 
PMID: 34059130; PMCID: PMC8165771.

27. Royal Marsden NHS Foundation Trust. Exploratory Study of Molecular 
Characterization in Patients With Metastatic Germ Cell Tumours 
Refractor y/Resistant to Platinum Treatment. CinicalTrials.gov 
identifier NCT03980587. Updated June 10, 2019. Accessed June 11, 
2023. https://clinicaltrials.gov/ct2/show/NCT03980587

28. Lavoie JM, Kollmannsberger CK. Current management of disseminated 
germ cell tumors. Urol Clin North Am.2019;46(3):377–388. doi: 
10.1016/j.ucl.2019.04.003. PMID: 31277732.

29. Murray MJ, Halsall DJ, Hook CE, Williams DM, Nicholson JC, 
Coleman N. Identification of microRNAs from the miR-371~373 and 
miR-302 clusters as potential serum biomarkers of malignant germ 
cell tumors. Am J Clin Pathol.2011;135(1):119–125. doi: 10.1309/
AJCPOE11KEYZCJHT. PMID: 21173133.

30. Gillis AJM, Rijlaarsdam MA, Eini R, Dorssers LCJ, Biermann K, Murray 
MJ, et al. Targeted serum miRNA (TSmiR) test for diagnosis and follow-
up of (testicular) germ cell cancer patients: a proof of principle. Mol 
Oncol.2013;7(6):1083–1092. doi: 10.1016/j.molonc.2013.08.002. PMID: 
24012110; PMCID: PMC5528443.

31. Dieckmann KP, Radtke A, Geczi L, Matthies C, Anheuser P, Eckardt U, et 
al. Serum levels of microRNA-371a-3p (M371 Test) as a new biomarker 
of testicular germ cell tumors: results of a prospective multicentric 
study. J Clin Oncol.2019;37(16):1412–1423. doi: 10.1200/JCO.18.01480. 
PMID: 30875280; PMCID: PMC6544462.

32. Nappi L, Thi M, Lum A, Huntsman D, Eigl BJ, Martin C, et al. Developing 
a highly specific biomarker for germ cell malignancies: plasma 
miR371 expression across the germ cell malignancy spectrum. J Clin 
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31553692; PMCID: PMC7351323.

33. Lafin JT, Singla N, Woldu SL, Lotan Y, Lewis CM, Majmudar K, et 
al. Serum microRNA-371a-3p levels predict viable germ cell tumor 
in chemotherapy-naïve patients undergoing retroperitoneal lymph 
node dissection. Eur Urol.2020;77(2):290 –292. doi: 10.1016/j.
eururo.2019.10.005. PMID: 31699528; PMCID: PMC7756387.

34. Leão R, van Agthoven T, Figueiredo A, Jewett MAS, Fadaak K, Sweet 
J, et al. Serum miRNA predicts viable disease after chemotherapy 
in patients with testicular nonseminoma germ cell tumor. J 
Urol.2018;200(1):126–135. doi: 10.1016/j.juro.2018.02.068. PMID: 
29474847.

35. Haukeland University Hospital. MicroRNA as Markers in Testicular 
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8, 2022. Accessed June 11, 2023. https://clinicaltrials.gov/ct2/show/
NCT04914026?cond=NCT04914026

36. SWOG Cancer Research Network. A Study of miRNA 371 in Patients 
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37. Merker JD, Oxnard GR, Compton C, Diehn M, Hurley P, Lazar AJ, et 
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38. Fleischhacker M, Schmidt B. Pre-analytical issues in liquid biopsy – 
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39. Ellervik C, Vaught J. Preanalytical variables affecting the integrity of 
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doi: 10.1373/clinchem.2014.228783. PMID: 25979952.

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