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The Past and Future of Biomarkers  
in Testicular Germ Cell Tumors
Aditya Bagrodia,1 Siamak Daneshmand,2 Liang Cheng,3 James Amatruda,4  
Matthew Murray,5,6 John T. Lafin1

1 Department of Urology, University of Texas Southwestern Medical Center, United States, 2 Department of Urology, University of Southern California, Keck School of 
Medicine, United States, 3 Department of Pathology, Indiana University School of Medicine, United States, 4 Cancer and Blood Disease Institute, Children’s Hospital 
Los Angeles, Departments of Pediatrics and Medicine, Keck School of Medicine, University of Southern California, United States, 5 Department of Pathology, University 
of Cambridge, United Kingdom, 6 Department of Pediatric Hematology and Oncology, Cambridge University Hospitals NHS Foundation Trust, Cambridge, United Kingdom

Abstract

Testicular germ cell tumor (GCT) is the most common malignancy in 18- to 40-year-old men. Unlike most other 
cancers, GCT is frequently curable even when metastatic. These tumors can be classified histologically into seminoma 
and non-seminoma, which determines treatment. Therefore, successful treatment requires accurate diagnosis, 
classification, and monitoring. Serum tumor markers, including lactate dehydrogenase, α-fetoprotein, and β-human 
chorionic gonadotropin, aid in the classification and staging of GCTs. These markers therefore play a critical role 
in the decision-making process when managing GCT patients. However, there exist many scenarios in which these 
markers fail to perform adequately. This is particularly true in the case of seminoma, where only 10% to 15% will have 
elevated serum tumor markers. Non-specific elevation of these markers is also a common occurrence, complicating 
the interpretation of borderline positive results, particularly in follow-up. To bridge this gap in performance, next 
generation biomarkers are being investigated. In this review, we consider the role of conventional serum tumor 
markers in GCT management and discuss recent advances in the next generation of biomarkers, with a focus on 
circulating microRNAs. We discuss the value that circulating microRNAs could bring as an addition to currently 
used markers, as well as potential weaknesses, in GCT management.

Introduction

Testicular germ cell tumor (GCT) is the most common solid tumor in 18- to 40-year-old men, and accounts for 
more life-years lost than any other non-pediatric malignancy [1]. GCTs are histologically classified as seminoma or 
non-seminomatous GCT (NSGCT). Seminomas retain pluripotency markers and genotypically resemble primordial 
germ cells, their presumed cell of origin. NSGCT can be further subdivided based on differentiation into embryonic 
germ layers (teratoma), toward extraembryonic elements (choriocarcinoma, yolk sac tumor) or early embryonic 
elements (embryonal carcinoma) [2]. These classifications are not mutually exclusive: approximately 15% of NSGCT 
also contain seminomatous elements [3]. Identification of these elements is critical to determining optimal clinical 
management.

Serum tumor markers (STMs) can help to identify the presence of GCT, as well as the presence of particular 
components. Conventionally, 3 STMs have been used to monitor GCT: lactate dehydrogenase (LDH), α-fetoprotein 
(AFP), and β-human chorionic gonadotropin (β-hCG). STMs are particularly useful in NSGCT, 60% to 85% of 
which secrete detectable STMs (Table 1). However, these markers are less helpful in seminoma, in which only 10% to 

Key Words Competing Interests Article Information

Testis cancer, germ cell tumor, microRNA, 
teratoma, serum biomarker, serum tumor 
markers

None declared. Received on June 26, 2020 
Accepted on August 15, 2020

Soc Int Urol J. 2020;1(1):77–84

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mailto:Aditya.bagrodia%40utsouthwestern.edu?subject=SIUJ


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15% of cases have elevated markers, and AFP is absent 
by definition [4]. Further, markers often are normal in 
the case of early recurrence or low tumor burden, and 
non-specific processes may cause them to be elevated. 
Despite these limitations, STMs have proven integral 
in the diagnosis and monitoring of GCTs. This is 
evidenced by the current staging system including an 
S-stage classification, indicating the presence of these 
markers [5].

Although current markers play a critical role in the 
management of GCTs, there are numerous scenarios 
where they fail to provide adequate information. 
Perhaps most glaringly, 10% to 50% of men presenting 
with clinical stage I disease (any T, N0M0S0) with 
normal STMs post-orchiectomy will harbor occult 
disease [6]. Up to 30% of patients with clinical stage II 
disease (T any N1M0S0-1) will ultimately be found to 
have pathologic N0 disease [7]. There is currently no way 
to identify these patients at point of care.

In this review, we discuss currently used conventional 
STMs in the context of GCT diagnosis and management. 
We also examine the next generation of GCT markers, 
particularly circulating microRNAs (miRNAs, miR), 
which hold promise to fill the critical gaps left by 
currently conventional STMs [6].

Conventional Serum Tumor Markers
Lactate dehydrogenase
Lactate dehydrogenase (LDH) is a ubiquitously 
expressed enzyme in all cells that reversibly converts 
lactate to pyruvate [8]. Circulating LDH levels are 
associated with high cell turnover, and the level may 
be elevated in non-GCT malignancies, including 
lymphoma and renal cell carcinoma. Elevated LDH has 
also been reported in the case of cell lysis or injury, such 
as rheumatologic disorders, myocardial infarction, and 
other muscular disease [8]. Therefore, LDH is the least 

GCT-specific of the 3 conventional STMs. This problem 
is exacerbated by the fact that the enzymatic activity 
assay used to detect LDH varies considerably across 
laboratories. In part because of this variation, the precise 
half-life of LDH is unknown, but it is on the order of 
days, not hours [9]. Despite low specificity, LDH has 
relatively high sensitivity compared with other STMs: it 
is elevated in 40% to 60% of GCTs and may be the only 
positive marker in the case of seminoma, where as few as 
30% of patients will have LDH elevation [8].

α-Fetoprotein
AFP is composed of an α-globulin molecule and 
carbohydrate moiety [9]. AFP is normally produced by 
the fetal yolk sac and liver, and therefore post-pubertal 
serum levels are generally low (<12 ng/mL) [9,10]. A 
chemiluminescent sandwich enzyme assay is typically 
used to detect it, which may have different upper limits 
of normal parameters across laboratories [9]. The half-
life of AFP is estimated at 5 to 7 days [8].

AFP is produced in most yolk sac tumors and can also 
be detected in some patients with embryonal carcinoma 
or teratoma elements. Composition of the teratomatous 
element may play a role in AFP levels; in pure teratoma 
specimens, AFP may be produced by gastrointestinal or 
hepatic elements in the tumor [9]. AFP level is related 
to clinical stage in NSGCT, with patients with stage I 
disease showing AFP elevation in 10% to 20% of cases 
before orchiectomy, and patients with metastatic disease 
showing elevation in 40% to 60% of cases [8].

Elevated AFP levels preclude a diagnosis of seminoma 
by definition [8]. Even if pathologic examination of the 
orchiectomy specimen reveals seminoma exclusively, 
very high levels of AFP indicate the presence also of 
an element of NSGCT. In exceptional cases AFP may 
be elevated in patients with seminoma with hepatic 
metastases when liver regeneration is occurring [11]. 
Minimal or non-specific AFP elevations must be 
considered with caution when attempting to classify 
such cases. Several reports exist of marginal AFP 
elevation (generally < 20 ng/mL) in cases of pure 
seminoma [12]. False positive AFP levels are often 
associated with certain liver conditions, including 
chronic liver disease, hereditary ataxia telangiectasia, 
hepatocellular carcinoma, or with a history of gastric or 
hepatic surgery [13]. Other potential scenarios include 
lung or gastrointestinal cancers, including colon, 
stomach, and pancreatic cancers [8]. Additionally, liver 
damage due to systemic therapy, alcoholism, or viral 
infection may lead to erroneously high AFP levels [14]. 
Therefore, absent other indications of GCT, it is 
recommended that patients with mildly elevated but 
stable AFP be managed by surveillance [10].

Abbreviations 

AFP α-fetoprotein
ß-hCG ß-human chorionic gonadotropin
GCNIS germ cell neoplasia in situ
GCT germ cell tumor
IGCCCG  International Germ Cell Cancer  

Collaborative Group
LDH lactate dehydrogenase
miRNA, miR- microRNA
NSGCT non-seminomatous germ cell tumor
RPLND retroperitoneal lymph node dissection
STM serum tumor marker

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β-Human chorionic gonadotropin
Hu m a n  c hor ion ic  gon a dot ropi n  ( hC G)  i s  a 
heterodimeric glycoprotein, composed of an α- and a 
β-subunit. Generally, both the α-β heterodimer and 
the free β-subunit are measured and combined into 
total hCG, referred to as β-hCG [9]. β-hCG is generally 
measured with a double antibody immunometric assay, 
with normal levels determined as < 2 IU/L [8]. Elevated 
β-hCG is found in both seminoma and NSGCT and 
is the most commonly elevated STM in adult GCT 
patients.

Approximately 15% to 20% of seminoma cases 
will have elevated β-hCG due to the presence of 
syncytiotrophoblast cells [8]. Although elevated β-hCG 
levels are associated with tumor bulk, there is no known 
association between β-hCG levels and risk of metastasis 
following orchiectomy in patients with stage I disease. 
β-hCG is also not incorporated into International 
Germ Cell Cancer Collaborative Group (IGCCCG) risk 
stratification for patients with pure seminoma [5].

Approx imately 10% to 20% of patients w it h 
stage I NSGCT w i l l have elevations in β-hCG 
before orchiectomy [8]. Up to 40% of patients with 
disseminated disease will display elevated β-hCG levels. 
However, β-hCG is detectable in a subtype-specific 
manner; although β-hCG can be present in patients 
with embryonal carcinoma, β-hCG levels are greatest in 
patients with significant portions of choriocarcinoma. 
In contrast to seminoma, the IGCCCG risk stratification 
criteria include β-hCG as an important prognostic factor 
for NSGCT [5].

Although β-hCG is the most frequently elevated 
STM in GCT, its specif icity remains a concern. 
Hypogonadism is a particular confounding factor, as 

it can occur following orchiectomy [8]. Hypogonadism 
leads to a compensatory increase in hCG from the 
pituitar y gland. Additionally, hy pogonadism can 
cause an increase in levels of luteinizing hormone 
(LH), which has an identical α- subunit and similar 
β-subunit to hCG. This can lead to a cross-reaction in 
the β-hCG assay and erroneous results, although most 
β-hCG assays no longer cross-react with LH. Additional 
potentially confounding factors include marijuana use, 
tumor lysis pursuant to chemotherapy, and the presence 
of heterophile antibodies [15,16]. Other cancer types, 
including lymphoma, leukemia, and neuroendocrine 
tumors, are also known to produce detectable β-hCG, 
but generally not to the high levels that can occur in 
some patients with GCTs (>10 000 IU/L) [15].

Serum microRNAs as Biomarkers in 
Testicular GCTs
Conventional STMs play an essential role in the 
diagnosis and monitoring of patients with GCTs. 
However, as outlined above, they are hampered in part 
by histological considerations and limited performance 
characteristics [6]. GCT-specific serum microRNAs 
(miRNA, miR-) [17–20] have emerged over the past 
decade as highly accurate tools for monitoring GCT 
patients, outperforming conventional STMs [9,21]. The 
measurement of these miRNAs may significantly change 
the way that GCT patients are diagnosed and treated.

MicroR NAs are short, non-coding R NAs that 
control gene expression in a target-specific manner [6]. 
Although longer RNA strands are notorious for being 
very labile, miRNA are remarkably stable, and have 
been proposed as markers in other cancer types [22,23]. 
In 2006, Voorhoeve et al. described overexpression of 
miR-372 and -373 in GCT tissues and cell lines, and 

TABLE 1.

Histology-specific serum AFP and β-hCG levels [9]

Histologic subtype AFP β-hCG

Seminoma – ±

Embryonal carcinoma ± ±

Choriocarcinoma – ++

Yolk sac tumor ++ –

Teratoma ± –

++: strongly positive levels; – : negative levels; ± : marker may be negative or moderately positive.  
Adapted from Murray MJ, Huddart RA, Coleman N.[9] with permission of Springer Nature.

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hypothesized that these miRNAs acted as oncogenes by 
inhibition of p53 signaling via LATS2 suppression [24]. 
A body of work following this study has since 
demonstrated overexpression of 8 miRNAs in malignant 
GCT tissues, all from the miR-371~373 and miR-302/367 
clusters [25,26]. This expression pattern was absent in 
teratoma, but otherwise occurred regardless of histology, 
site of origin, or sex or age of the patient. Critically, 
these results were the first to demonstrate a universal 
molecular abnormality of this disease.

In the next seminal study in this arena, Murray 
and colleagues demonstrated that through the use 
of a highly sensitive qPCR-based assay, including a 
pre-amplification step, these malignant GCT-specific 
miRNAs were elevated in the serum of a GCT patient 
compared with a pool of normal controls [27]. The 
levels were also found to be informative of treatment 
response and disease status on follow-up. These results 
triggered an avalanche of investigations concerning 
the performance and potentia l inclusion of the 
serum miRNA test in routine clinical diagnosis and 
management for patients with GCTs.

Screening
There are few data available to assess whether GCT-
associated miRNAs are useful in screening for the 
development of invasive GCT. However, there may be 
some utility of serum miRNAs in the case of screening 
at-risk patients for the presence of germ cell neoplasia 
in situ (GCNIS), the presumed precursor lesion of post-
pubertal GCTs. Novotny et al. reported overexpression 
of GCT-associated miRNAs in GCNIS tissue compared 
with normal adult testis controls [28]. An initial study 
suggested that serum miRNAs do not detect GCNIS, 
but a subsequent study indicated that up to 50% of 
patients with GCNIS lacking frank GCT have elevated 
serum miRNAs [29,30]. Importantly, these studies were 
conducted in a small number of patients and further 
validation is required in this setting.

Pre-orchiectomy
A solid testicular mass detected by physical examination 
or sonography is initially considered a malignancy. 
These techniques can overlook non-malignant causes 
of a testicular mass, and therefore STMs may play an 
important role here. Conventional STMs may not be 
informative in this context, particularly in seminoma, 
where only 10% to 15% of cases show elevated 
markers [4]. The superior sensitivity of circulating 
miRNAs over conventional STMs could therefore be 
valuable in the pre-orchiectomy setting.

This setting is where the utility of circulating miRNAs 
has so far been best described (Table 2). In 2013, Gillis 
et al. examined serum miRNA expression in 80 GCT 
patients, in non-cancer controls, and in patients with 

non-GCT testicular masses [31]. Using magnetic bead 
purification but no pre-amplification step, this study 
identified that a 4-member panel (miR-371a-3p, miR-
372-3p, miR-373-3p and miR-367-3p) outperformed 
a full panel that included miR-302a~d from the miR-
302/367 cluster, refining the necessary targets for an 
informative test. The test performance was acceptable, 
with 98% sensitivity and 48.3% specificity, although 
without the pre-amplification step [28], specificity was 
necessarily low in order to retain high sensitivity. The 
test was negative for non-GCT testicular masses and 
returned to normal in post-orchiectomy stage I GCT 
patients. This revised 4-member panel would set the 
basis for future studies.

These results were confirmed in an expanded 
cohort by van Agthoven and Looijenga in 2017 [29]. 
Magnetic bead purification was used in combination 
with the standard pre-amplification step to examine 
a 3-member panel lacking miR-372-3p in 250 GCT 
patients, 60 non-GCT patients, and 104 healthy male 
controls. Performance improved as expected, with a 
90% sensitivity and 91% specificity in the combined 
panel. Importantly, this study aligned with previous and 
subsequent reports that serum miRNA does not detect 
pure teratoma [32–34].

More recently, attempts have been made to reduce the 
4-member panel further, while maintaining sensitivity. 
In 2017, Dieckmann et al. compared the performance 
of circulating miR-371a-3p to the full 4-member 
panel, using a column-based extraction and standard 
pre-amplification. This study reported comparable 
performance between miR-371a-3p alone and the full 
panel (92% sensitivity, 85% specificity) [34]. Following 
this report, Dieckmann et al. used the same method to 
examine miR-371a-3p exclusively in 616 GCT patients 
and 258 controls, the largest cohort yet [35]. This study 
reported 90% sensitivity and 94% specificity in viable 
GCT, a return to normal levels following orchiectomy 
in the majority of stage I patients, and no detectable 
circulating miR-371a-3p in pure teratoma. No long-term 
follow-up data were available to determine the clinical 
significance of persistently elevated serum miR-371a-
3p levels post-orchiectomy in clinical stage I patients, 
which is an outcome measure in current clinical trials 
(see below) [20].

The refinement of target selection and methods in the 
context of pre-orchiectomy has resulted in a miRNA-
based test that significantly outperforms conventional 
STMs. Although these methods have begun to converge 
to a single standard of normalization and quantification, 
further work is required. These refined methods are now 
being used in other scenarios, some examples of which 
follow.

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Identification of occult metastases in patients 
with early stage I/stage I disease
From 10% to 50% of patients with clinical stage I viable 
GCT, normal conventional STMs, and no radiographic 
evidence of metastasis will ultimately relapse [6]. It is 
extremely challenging to identify which of these patients 
harbor occult disease, as diagnostic tools are limited. 
Further, 85% to 90% of stage IA NSGCT patients will be 
cured by orchiectomy alone; surveillance is preferred in 
these patients to prevent overtreatment, but the potential 
for metastases currently cannot easily be ruled out [36]. 
Patients with occult disease would be better served if 
they were to receive a single cycle of BEP or primary 
retroperitoneal lymph node dissection (RPLND), as both 
are associated with excellent outcomes [37]. Additionally, 
early identification of occult metastatic seminoma could 
permit earlier, less intensive treatment.

Circulating miRNA levels are associated with tumor 
mass, raising potential concerns about whether they 
reach detectable levels in the case of radiographically 
occult metastases [34]. Recent studies provide evidence 
that circulating miRNA levels are detectable even 
when current identification strategies fail. Nappi et al. 
reported that miR-371a-3p levels in plasma accurately 
predicted relapse in all cases in their small cohort [38]. 
Lafin et al. demonstrated that serum miR-371a-3p 
accurately detected minimal residual pathologically 
confirmed viable GCT at primary RPLND [39]. In this 
24-patient cohort with normal conventional STMs, miR-
371a-3p showed a 100% sensitivity and 92% specificity, 
demonstrating the value of circulating miRNAs in this 
setting.

These studies and others suggest that circulating 
miRNAs may help avoid overtreatment of the 20% 
to 30% of stage IIA, marker negative patients that are 

ultimately devoid of occult disease on histology [7,40]. 
Additionally, because low circulating miRNA levels 
immediately before chemotherapy are associated 
with complete response, this tool may in the future 
aid clinicians deciding between obser vation and 
further treatment [36]. Similarly, this principle might 
be extended to patients who have received primary 
RPLND and who are being considered for surveillance 
or adjuvant chemotherapy.

Response to treatment in patients with 
disseminated disease
The utility of circulating miRNAs in the context of 
chemotherapy is an area of active study. Dieckmann 
et al. found in patients with stage II/III GCT that serum 
miRNA levels both tracked treatment response during 
chemotherapy and, in 2 cases, suggested resistance [35]. 
Seventy out of 118 patients with systemic disease 
exhibited a decrease in serum miR-371a-3p levels after 
the first cycle of chemotherapy. As a group, stage II 
patients exhibited no further statistically significant 
reduction after the first cycle, while stage III patients 
ex hibited no further reduction af ter the second 
cycle. The 2 patients in this cohort who experienced 
disease progression and ultimately died showed rising 
circulating miR-371a-3p levels. Mego et al. examined 
plasma miRNA levels in 180 patients with metastatic 
GCT treated with systemic therapy and found that 
miRNA levels were associated with IGCCCG risk group 
and response [41].

Patients with primary metastatic or relapsed GCT 
will receive induction chemotherapy, and those without 
radiographically complete response will receive a post-
chemotherapy RPLND [41]. This surgery is currently the 
only accurate method to determine if residual masses 
harbor teratoma or viable GCT. It is also the only way 

TABLE 2.

Performance characteristics of serum pre-orchiectomy GCT-associated miRNAs for prediction of GCT on final 
orchiectomy histopathology.

Reference Sensitivity, % Specificity, % PPV, % NPV, % AUC

Gillis et al. [27] 98 48.3 NR NP 0.96

Van Agthoven and 
Looijenga [26]

90 91 94 7 0.962

Dieckmann et al. [31] 90.1 94 97.2 82.7 0.966

AUC: area under the curve; GCT: germ cell tumor; NPV: negative predictive value; PPV: positive predictive value.

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to treat teratoma and can contribute to therapeutic 
management in patients with chemo-resistant viable 
GCT [42,43]. However, approximately 50% of patients 
receiving this operation will harbor only fibrosis/
necrosis, meaning 50% of the patients who undergo this 
operation will have done so unnecessarily. Leão et al. 
examined circulating miRNA at post-chemotherapy 
RPLND and found that miR-371a-3p exhibited the best 
performance to detect residual viable GCT, but did not 
find any discriminatory capacity for the presence of pure 
teratoma [44]. A recent report from TCGA expression 
data suggested that teratoma and yolk sac tumor 
tissue express high levels of miR-375 [45]; however, 
Lafin et al. did not find any discriminatory capacity of 
this circulating miRNA in 3 teratoma-only patients at 
RPLND [39]. Additional studies have confirmed these 
negative results in larger cohorts [42,43].

Surveillance
Circulating miRNAs represent a powerful tool capable 
of changing the way GCT patients are monitored. 
Because this assay cannot detect pure teratoma, the 
likelihood of teratoma formation must always be kept 
in mind. The development of teratoma is an extremely 
rare occurrence in patients with pure seminoma, 
and in this arena, circulating miRNAs may in future 
supplant axial imaging and conventional STMs. For 
patients with NSGCT, teratoma formation must remain 
a consideration and warrants ongoing infrequent axial 
imaging. In this setting, circulating miRNAs may offer 
a complementary test, permitting a reduction in the 
number of axial scans required for surveillance [6].

Conclusions
Conventional STMs play a critical role in the current 
diagnosis, treatment, and monitoring of patients with 
GCT. Unfortunately, their performance is limited in 
many situations by false positives and low sensitivity. 
This limitation extends from diagnosis, through 
treatment and into surveillance, impacting decision-
making across the spectrum of patient care. The promise 
of circulating miRNAs as an addition to these markers 
is supported by a growing body of literature [17–20, 
27,29,35]. Inclusion of circulating miRNAs alongside 
conventional STMs could aid in identification of false 
positives, such as in a case report describing elevated 
AFP levels pursuant to liver regeneration [12]. Two 
large clinical trials (AGCT1531 [NCT03067181] and 
SWOG-S1823) are further studying the role of miRNA 
in patients with GCTs.

Despite generally excellent performance of circulating 
miRNAs, limitations remain. First, a standard method 
of collection, normalization, and quantification must 
be devised to reduce variation across laboratories and 
enable large-scale validation. Second, identification of 
pure teratoma by circulating biomarkers remains an 
elusive target. Differentiation of pure teratoma from 
viable GCT is an unmet clinical need; recommendation 
of surgery or chemotherapy will often depend on 
segregating the two.

Despite t hese modest limitations, circu lating 
miRNAs have the potential to change the way that GCT 
cases are managed, aiding clinicians in decision-making 
to provide greater benefit to their patients.

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