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EXPERIMENTAL AND THERAPEUTIC MEDICINE 22: 1479, 2021
Abstract. Neuroendocrine neoplasms (NENs) are a
heterogeneous group of rare tumors with different types of
physiology and prognosis. Therefore, prognostic informa‑
tion, including morphological differentiation, grade, tumor
stage and primary location, are invaluable and contribute to
the formulation of treatment decisions. Biomarkers that are
currently used, including chromogranin A (CgA), serotonin
and neuron‑specific enolase, are singular parameters that
cannot be used to accurately predict variables associated with
tumor growth, including proliferation, metabolic rate and
metastatic potential. In addition, site‑specic biomarkers, such
as insulin and gastrin, cannot be applied to all types of NENs.
The clinical application of broad‑spectrum markers, as it is the
case for CgA, remains controversial despite being widely used.
Due to limitations of the currently available mono‑analyte
biomarkers, recent studies were conducted to explore novel
parameters for NEN diagnosis, prognosis, therapy stratica‑
tion and evaluation of treatment response. Identication of
prognostic factors for predicting NEN outcome is a critical
requirement for the planning of adequate clinical manage‑
ment. Advances in ‘liquid’ biopsies and genomic analysis
techniques, including microRNA, circulating tumor DNA or
circulating tumor cells and sophisticated biomathematical
analysis techniques, such as NETest or molecular image‑based
biomarkers, are currently under investigation as potentially
novel tools for the management of NENs in the future. Despite
these recent ndings yielding promising observations, further
research is necessary. The present review therefore summa‑
rizes the existing knowledge and recent advancements in the
exploration of biochemical markers for NENs, with focus on
gastroenteropancreatic‑neuroendocrine tumors.
Contents
1. Introduction
2. Aim and search strategy
3. Currently available biomarkers
4. Potential novel biomarkers
5. Conclusions
1. Introduction
Neuroendocrine neoplasms (NENs) are a heterogenous group
of rare malignancies that arise from neuroendocrine cells
distributed throughout the body and produce peptide hormones
and/or biogenic amines (1). NENs can be dichotomised into
‘functional’ (F) and ‘non‑functional’ (NF) tumors (2). The
F‑NENs category represents 33% of all NENs and is mainly
characterized by well‑defined clinical symptoms that are
caused by the over secretion of their circulating products (2).
Examples of F tumors include NENs in the midgut (such as the
small intestine), which are classically associated with carci‑
noid syndrome (CS) due to the overproduction of serotonin (3).
Symptoms of CS have been previously reported to be asso‑
ciated with the overproduction of serotonin by F NENs. By
contrast, NF NENs are more common in terms of incidence
and can cause mechanical symptoms, including ischemia or
obstruction, the complications of which from local tumor
growth can ultimately result in mortality (2).
Due to the substantial heterogeneity in these NENs both
in terms of clinical aggressiveness and response to therapy,
management of patients with such diseases is a signicant
challenge (4). In total, 60‑80% of NENs have already metasta‑
sized on diagnosis (5). Prognostic and predictive markers have
been intensively investigated to explore the optimal clinical
management strategy for this category of neoplasms (6).
Investigation into prognostic markers contribute valuable
information in understanding the physiology of NENs and their
natural course. They reveal benecial mechanistic information
underlying the aggressive properties of the disease and the risk
of recurrence or death. By contrast, predictive markers can be
used to estimate the benet of a certain therapy compared with
their corresponding condition at baseline, which can assist
in implementing the optimal treatment strategy for the most
favorable outcome (7).
Perspectives on the diagnostic, predictive and prognostic
markers of neuroendocrine neoplasms (Review)
OANA ALEXANDRA CIOBANU1,2, SORINA MARTIN1,2 and SIMONA FICA1,2
1Department of Endocrinology and Diabetes, Elias Hospital, 011461 Bucharest; 2Department of Endocrinology,
Carol Davila University of Medicine and Pharmacy, 20021 Bucharest, Romania
Received July 27, 2021; Accepted September 23, 2021
DOI: 10.3892/etm.2 021.10914
Correspondence to: Mrs. Oana Alexandra Ciobanu, Department
of Endocrinology and Diabetes, Elias Hospital, 17 Marasti Avenue,
Sector 1, 011461 Bucharest, Romania
E‑mail: oana‑alexandra.ciobanu@drd.umfcd.ro
Key words: neuroendocrine neoplasms, biomarkers, prognostic
markers, liquid biopsies, molecular imaging
CIOBANU et al: CURRENT AND POTENTIAL MARKERS IN NEUROENDOCRINE NEOPLASMS
2
At present, the most important prognostic factor for NENs
is morphological differentiation characteristics, which can
be divided into the well‑ or poorly‑differentiated categories,
corresponding to neuroendocrine tumors (NETs) or neuroen‑
docrine carcinomas (NECs) (8). Furthermore, NENs display
a varying degree of proliferation known as grade (G), which
is approximated using the Ki‑67 proliferation index and
mitotic count (9). Specically, the latest 2019 World Health
Organization (WHO) classication of gastro‑entero‑pancre‑
atic (GEP)‑NENs distinguishes three grades of NENs that are
classied into the low (G1), intermediate‑(G2) and high‑grade
(G3) categories based on the degree of differentiation, with
the mention that poorly differentiated NECs are considered
denitively G3 (10,11). Although the WHO classication of
tumors is considered to be the gold standard tumor classica‑
tion, it differs depending on their primary site in the body. For
example, the 2015 WHO Classication of Tumors of the Lung,
Pleura, Thymus and Heart does not make use of the Ki‑67
proliferation index (11). However, the current consensus is to
improve the management of NENs by adopting an uniformized
nomenclature system towards different organs (12). It is hoped
that this new classication will serve as a novel grading tool
that can be introduced into common clinical practice to reveal
essential information regarding NENs (12).
In addition to adequate classication and grading, tumor
staging also carries prognostic signicance (13). European
Neuroendocrine Tumors Society (ENETS) and tumor, nodes
and metastases (TNM) classication of the American Joint
Committee on Cancer (AJCC) are currently used staging
systems that present the classification criteria but are not
identical and are site specic (14,15). A number of studies
have reported comparative data between the two systems
for the classification of pancreatic neuroendocrine tumors
(Pan‑NETs), suggesting a similar prognosis being found
by the two systems in ENETS and TNM of the AJCC, both
regarding progression free‑survival (PFS) and overall survival
(OS) (16,17). However, further studies are needed, as the
ENETS staging system appeared to be superior in stratifying
prognosis for each stage of pancreatic NENs according to a
previous report (18).
However, in terms of the formulation of concrete guide‑
lines for the clinical management of these tumors, numerous
issues persist due to the lack of comprehensive databases and
registries (9). In addition, an insufcient number of lesion
types have been studied despite wide variations in tumor
heterogeneity (9). The 5‑year survival rate ranges between 15
and 95%, depending on the location of the tumor primary site,
the level of metastatic spread at diagnosis, the available treat‑
ment options and the geographical site of care (19‑21).
Several methods have been reported to predict OS and
PFS, including nomograms taking into account the number
of liver metastases, tumor size and the Ki‑67 index (22), the
blood neutrophil‑lymphocyte ratio, Ki‑67 index and the lymph
node ratio (23) or phosphorylated histone H3 (24). However,
further research is required.
An important obstacle in clinical practice is the scarcity
of a set of sensitive and specic tumor biomarkers (25). The
‘perfect’ biomarker would ideally be characterized by high
sensitivity in diagnosing NENs, good prediction of disease
evolution and response to therapy (26). Currently available
NETs biomarkers belong to the mono‑analyte class and vary
in the rates of sensitivity and specicity for indicating the
biological characteristics, such as primary tumor site, or
functional or non‑functional type secretion. In addition,
these mono‑analyte measurements are unable to dene the
state of disease progression or efcacy of therapy (27), which
frequently do not correlated well with radiological evaluation
data (28). To overcome these shortcomings, interest in the
molecular profiling of NENs is increasing. Recent studies
have demonstrated the utility of molecular imaging as a
viable option for predicting the prognosis of NENs (29,30).
Since the density of somatostatin receptors (SSTRs) can also
be quantitively assessed using immunohistochemistry (IHC)
in the surgical specimens, this can be used to predict treat‑
ment response (31‑33). Furthermore complementary imaging
modalities or early radiological biomarkers, such as the tumor
grade rate (TGR), have been proposed to contribute valuable
prognostic information (34,35).
Molecular diagnostics have previously been performed
on biopsies of solid tumor tissues (13). As one branch of
this technique, ‘liquid’ biopsy is a non‑invasive strategy that
can provide the opportunity to investigate the molecular
genomic mechanism of tumor cells in circulating blood (the
‘tumor circulome’) to characterize the circulating molecular
proles, which may be useful for subsequent diagnosis and
monitoring (36,37). Emerging biochemical and therapeutic
markers have become increasingly popular since 2004 and
their relevance are expected to increase exponentially by
2050 (13,38,39). Identifying novel parameters is important for
optimally predicting the prognosis and treatment response for
each patient. Therefore, the present review summarizes some
of the current and future directions in this eld of research
with an emphasis on GEP‑NENs.
2. Aim and search strategy
The aim of the present review is to summarize the available
data on the diagnostic, prognostic and predictive biomarkers
for NENs. The review is structured into the following two
parts: i) Discussion of the markers most commonly used in
clinical practice at present; and ii) potential future diagnostic,
prognostic and predictive biomarkers.
Search strategy. Published data in the SCIENCEDIRECT
(https://www.sciencedirect.com/) and PUBMED
(https://pubmed.ncbi.nlm.nih.gov/) databases were collected
and analyzed. Publications from the past 5 years were
prioritized and selected using the following relevant key words
either alone or in combination: ‘Neuroendocrine neoplasms’,
‘neuroendocrine tumors’, ‘biomarkers’, ‘liquid biopsies’ and
‘molecular imaging’. Additional studies were identied by
reviewing the references of all selected articles, whereas publi‑
cations from major scientic meetings were searched manually.
After the exclusion of 52 duplicates, 353 articles were scanned,
following which 99 titles were removed because they didn't
satisfy the subject of the present review. We included A total
of 254 publications were selected for a full‑text evaluation.
The inclusion criteria were clinical and practical relevance in
the management of NENs. Full articles and English language
published original and review papers were included that
EXPERIMENTAL AND THERAPEUTIC MEDICINE 22: 1479, 2021 3
addressed the diagnosis and especially the prognostic and
predictive markers in NENs, with different degrees of statis‑
tical power due to the rarity of these neoplasms. Exclusion
criteria were case reports and publications with abstracts not
relevant for the present review were removed (case studies
removed, four; publications removed, 129), so that at the end of
the selection process, 121 publications were included (Fig. 1).
3. Currently available biomarkers
A number of biomarkers are currently applied for the diag‑
nosis of NENs. However, they are frequently not correlated
with diagnosis or clinical outcomes, mainly due to sensitivity
and specicity issues (Table I).
Chromogranin A (CgA). Human CgA is a glycoprotein that
belongs to the family of chromogranins and is localized
in the secretory granules alongside peptide hormones and
catecholamines throughout the neuroendocrine system (40).
Recognition of CgA as the default NET biomarker is widely
applied over the past decade due to its broad‑spectrum nature,
with high levels being found in both F and NF NENs (41‑43).
Yet, CgA detection and reliability are still based on different
non‑standardized assays (44). However, Cg A is highly
expressed in NETs tissue, but can also be measured in serum
or plasma as a widely used circulating tumor marker (40). High
levels of CgA are mainly observed in well‑differentiated NETs
and are associated with larger tumor burden, especially in gut
NETs (5,45). By contrast, CgA suffers from low sensitivity
for poorly‑differentiated tumors, where its production is less
pronounced (40,46).
As a result, enthusiasm for the application of CgA as a
marker is waning, which is compounded by the accumu‑
lating evidence of its low utility (43,47). The specic cut‑off
value (identified by the receiver operating characteristic
analysis between different assays) (44), primary location of the
NEN (48), endocrine‑associated syndrome (5), disease spread,
liver metastases (49), false‑positive elevations in CgA typi‑
cally caused by proton pump inhibitors, atrophic gastritis and
kidney failure (13), can all inuence the accuracy of this test.
However, a recent systematic review and meta‑analysis
evaluated the role of CgA in bronchopulmonary NENs because
of the scarcity of evidence in this eld (43). This previous
study reported the clinical utility of CgA for the diagnosis of
lung NENs, especially in small cell lung cancer (SCLC) with
a mean diagnostic specicity of 79.5±3.1 and sensitivity of
59.9±6.8%. Still, this nding require further validation (43).
Urinary 5‑hydroxyindoleacetic acid (5‑HIAA). 5‑HIAA is
a metabolite of serotonin, which is excessively produced by
serotonin‑secreting tumors and is excreted in the urine (13).
Prior to interpretating the 5‑HIAA test measurement in the
urine as a result, pharmacological and dietary artifacts must
rst be ruled out, typically by avoiding the intake of trypto‑
phan‑ and serotonin‑rich foods (50). Measurement of urinary
5‑HIAA excretion is more practical for patients with primary
midgut (jejunoileal, appendiceal and ascending colon) NETs,
which produces the highest levels of serotonin (51). For carci‑
noid syndrome (CS), this test has a reported sensitivity of >90%
and a specicity of 90% (52). Additionally, a level of 5‑HIAA
>300 mmol/24 h and three ushing episodes per day can be
considered to be a predictive factor of carcinoid heart disease
(CHD) (53). There is also evidence that 5‑HIAA in combination
with N‑terminal probrain natriuretic peptide (NT‑proBNP) can
be accurately used in screening for CHD (54). Nevertheless, its
prognostic role remains controversial.
N‑terminal Pro‑Brain Natriuretic Peptide (NT‑proBNP).
NT‑pro BNP is a peptide that is released by myocardial cells
as a result of an increase in heart volume and pressure and is
used especially for CHD prediction, which has been previously
correlated with patient survival (55,56). A high sensitivity
and specicity for CHD, 92 and 91%, respectively, has been
reported at the cut‑off value of 260 pg/ml (57). In addition,
measurement of the level of serum NT‑proBNP has been
recommended for intestinal NENs and CS for the diagnosis
and follow‑up of CHD (54).
Neuron specic enolase (NSE). NSE is the main enolase‑isoen‑
zyme present in neuronal and neuroendocrine tissues (58).
Although mainly expressed poorly‑differentiated NET cells,
measurement of serum NSE cannot be used to distinguish
among different subtypes of NENs (59). Consequently, although
elevated levels of NSE seems to have a degree of prognostic
value for poor outcome and correlate with tumor burden, NSE
is now rarely used in clinical practice since it is inferior to
CgA in terms of the information that can be extracted (59).
Figure 1. Flow chart for the selection of studies discussed in the present
re vie w.
CIOBANU et al: CURRENT AND POTENTIAL MARKERS IN NEUROENDOCRINE NEOPLASMS
4
However, NSE is currently applied as the default tumor marker
for SCLC diagnosis, prognosis and follow‑up, even though
elevated levels of NSE can also be found in non‑small cell lung
cancer (NSCLC) (58).
Pancreatic polypetide (PP). PP is serum marker produced by
the neuroendocrine cells of the colon and pancreas (60). PP
is considered to be a non‑specic marker for NENs, where
its levels has a modest degree of accuracy as a diagnostic
marker (13). However, it appears that an >50% increase in the
serum levels of PP in GEP‑NETs patients has a direct propor‑
tional relationship with the increase in tumor according to
RECIST 1.1 criteria on conventional imaging (CT scan ± MRI
scan) imaging (60).
Connective tissue growth factor (CTGF). CTGF is a modular
secreted protein that serves an important role in complex
biological mechanisms, including angiogenesis, tumorigen‑
esis and wound healing (61). In addition, CTGF can regulate
various types of brosis formation, such as cardiac brosis (62).
Therefore, CTGF has a reported sensitivity of 80% in detecting
heart disease (62). For NETs, previous studies have reported
that CTGF expression measured in plasma is more likely to
be found in small‑bowel ileal rather than in bronchial, pancre‑
atic or rectal NETs (63,64). However, a high level of CTGF
was found to be an independent predictive factor for right
ventricular dysfunction (61).
Ki‑67 proliferation index /mitotic count. Mitotic count,
Ki‑67 index and necrosis are typically used for the grading
of NENs (9). While mitotic count is recommended to be
reported as mitoses per mm2 area, in clinical practice this
process could be affected by limited areas available for
counting. On the other hand, Ki 67 is determined using IHC
measured in the most mitotically active areas of the patho‑
logical specimen, but due to the intratumoral heterogeneity
this process can be affected (9). However, this manner of
scoring can be time‑consuming, which is compounded by
the lack of consensus in the optimal method for determining
the proliferative rate (9,65). In clinical practice, when there
is a discordance between these two types of measurements
in assigning the grade, higher ‘grade counts’ are normally
negatively associated with poorer prognosis (65‑67). For
example, in ~33% of well‑differentiated Pan‑NETs, a G1
grade is determined based on the mitotic count, whereas
the G2 grade is determined using the Ki‑67 prolifera‑
tion index (66). In addition, the same percentage (33%) of
G2 Pan‑NETs are found for after mitotic count, but G3
according to the Ki‑67 proliferation index (64). Therefore,
the use of Ki‑67 IHC staining is essential for accurately
grading well‑differentiated NETs (65,66)
T h e lat e s t 2 019 W H O cl a ss i c a ti o n of GEP‑ N E Ns (10,67 )
now recognizes the category of high‑grade tumors as those
with a Ki‑67 index of >20%, of which well‑differentiated
GEP‑NENs represent ≤7% of all NENs (68). In addition,
tumor grade based on the Ki‑67 index cut‑off values has been
demonstrated to correlate with patient survival independent
of tumor stage in primary and metastatic GEP‑NENs (69,70)
In particular, the Ki‑67 index has also been considered
to be a viable prognostic marker for recurrence after the
resection of PanNETs (71). In liver metastases, the Ki‑67
index has a predisposition to be higher compared with that
in primary GEP‑NETs, where a proportional relationship
between the size of the metastasis and the Ki‑67 index was
found (72‑74)
In clinical practice, detection of multiple primary
tumors, multiple lymph node metastases and/or multiple
distant metastases are recommended for evaluating tissue
blocks with the biggest focus of tumor due to the intratu‑
moral heterogeneity (75). Furthermore, the Ki‑67 index
may present a variability during the course of the disease or
between primary tumor and metastasis (76). In a previous
study, the Ki‑67 index was evaluated in 103 GEP‑NETs, of
which 24% presented with higher grades in the metastasis,
10% with higher grades in the primary tumor and 66%
with same grade between the metastasis and the primary
tumor (77). The PFS and OS were found to be identical for
both G1 primaries and G2 metastasis categories, and also
for G2 primaries and metastasis; however, these are worse
compared with the G1 primary tumors only (G1 stable
category). Therefore, any G2 tumors (in primary or in
metastases of NETs) inuences patient survival.
The Ki‑67 index also hold promising potential for
clinical application in NECs, since a Ki‑67 index with a 55%
cut‑off for response was found to be associated with poorer
prognosis, favorable response to platinum‑based chemo‑
therapy (CHT) (78) and adverse reactions to temozolomide
CHT (79).
Table I. Sensitivity and specicity of current biomarkers.
First author/s, year Tumor marker Primary tumor location Sensitivity % Specicity % (Refs.)
Oberg et al, 2015 Chromogranin A Nonspecic 60‑90 <50 (2)
Feldman, 1986 Urinary 5‑hydroxyindole Midgut 70 90 (51)
acetic acid
Bhattacharyya et al, 2008 N‑terminal probrain Midgut: carcinoid heart 92 91 (57)
natriuretic peptide disease
Baudin et al, 1998 Neuron specic enolase Nonspecic 33 68 (59)
Walter et al, 2012 Pancreatic polypeptide Pancreas No data 84 (60)
Laskaratos et al, 2017 Connective tissue Midgut: right ventricular 88 69 (61)
growth factor dysfunction
EXPERIMENTAL AND THERAPEUTIC MEDICINE 22: 1479, 2021 5
4. Potential novel biomarkers
The current demand in this eld is to improve the methodology
for the diagnosis, treatment and prognosis of NENs. Therefore,
novel markers have been evaluated over the past decade.
Genetic mutations. Recent studies have benefited from
advances in sequencing technology for characterizing the
complex molecular landscape of NENs (80,81). Regarding
PanNETs, the current trend is mainly focused on deter‑
mining the roles of multiple endocrine neoplasia 1 (MEN1),
death‑domain associated protein (DAX X), α‑thalassemia and
mental retardation syndrome X‑linked (ATR X) genes in the
alternative lengthening of telomeres (ALT) axis and compo‑
nents in the mTOR and DNA damage pathways (82,83). In
particular, molecular alterations have been consistently associ‑
ated with the following four events: DNA damage repair; cell
cycle regulation; PI3K/AKT/mTOR signaling; and telomere
maintenance (84).
DAXX and ATRX expression and ALT. Recent studies have
revealed increasingly consistent ndings despite the lower
mutation burden of PanNETs (82) as mutations in the MEN1,
ATRX and DA XX genes were frequently observed in this type
of tumors (85‑87). In total, up to 40% of well‑differentiated
NENs in the pancreas exhibit somatic mutations in the DAXX
and ATRX genes (82). These two genes encode proteins that
interact and have multiple cellular functions, including modu‑
lating telomeric chromatin. ATRX and DAX X interact to deposit
histone H3.3‑containing nucleosomes in the centromeric and
telomeric regions of the genome and may interact to suppress
the ALT pathway under normal circumstances (88,89).
ATRX/DAXX mutations result in loss of nuclear expression
of their proteins, as detected by IHC, in tumor tissue, which
correlates with the suppression of ALT (90). A negative
expression of ATRX/DAXX is typically associated with
well‑differentiated NENs, and correlate with worse survival
in Pan‑NETs (90,91). ALT is a telomerase‑independent
telomere maintenance mechanism that has been previously
studied using uorescence in situ hybridization (92). Altered
telomeres are a key process frequently found in PanNETs (93).
As such, positive ALT status in liver metastases of NENs was
found to associate with worse survival and increased risk of
recurrence (94,95). It has been previously shown that there is
100% concordance among the ALT phenotype, ATRX/DAXX
mutations and/or protein loss (92), where the presence of
ALT‑positive and ATRX/DAXX‑negative (inactivation muta‑
tion) in well‑differentiated PanNETs is associated with a
signicantly higher grade, size, grading, vascular/perineural
invasion, metastatic disease and with reduced relapse‑free
and tumor‑specic survival (96). Therefore, this prole can be
applied as a marker of more aggressive PanNET phenotypes
for patient stratication (97,98).
Application of fine‑needle aspiration (FNA) makes it
possible to detect the loss of ATRX/DAXX and the presence
of ALT as a non‑invasive method to sample tumors. As afore‑
mentioned, somatic mutations of ATRX/DAXX genes can be
detected using IHC to indicate a loss of nuclear expression of
their respective proteins (90). In addition, ALT can be assessed
using telomere‑specic uorescence in situ hybridization (92).
This procedure is becoming increasing important in clinical
practice, through which ALT or the loss of ATRX or DAX X
expression can be veried with higher degrees of condence
during the prognostic process (92).
mTOR signaling pathway. Somatic gene alterations are
currently studied in PanNETs (98,99), of which two processes
have been frequently observed to be affected chromatin
remodeling and activation of PI3K/Akt/mTOR signaling (100).
The mTOR pathway regulates cell proliferation, cell cycle
and apoptosis; however, in tumor cells, an abnormal activa‑
tion of mTOR pathway influences the tumor to grow and
metastasize (101). Since 14.7% of PanNET cases exhibit
mutations in the PI3K/Akt/mTOR pathway, this nding that
can be exploited to select patients for treatment with mTOR
inhibitors (100,102).
Mutations in the tuberous sclerosis complex 2 (TSC2) and
PTEN genes are suppressors of the Akt/mTOR pathway, which
are present in up to 11% of sporadic PanNETs (100). Reduced
expression of both PTEN and TSC2 was found to be associated
with more aggressive phenotypes, presence of liver metastases
and reduced disease‑free survival and OS in a cohort of 72
primary PanNETs according to microarray analysis (99).
However, further studies are warranted before mutations
of components in the PI3K pathway can be considered as a
biomarker of response.
Retinoblastoma protein 1 (RB1) and p53 in NECs but not in
NE Ts. The most recent 2019 WHO classication of GEP‑NENs
made a clear distinction between NET G3 and NEC (10). NET
G3 lacks consensus‑based recommendations is associated
with longer OS (98). In certain cases, histology is not suf‑
cient for differentiating between highly proliferative NETs
and NECs (103), where it is hoped that molecular proling can
amend this deciency. Abnormal immunolabeling of p53 and
RB1 pathways are currently being proposed for the distinction
of well‑differentiated NETs, especially NETs G3 from poorly
differentiated NECs during diagnosis (103). Although p53
and RB1 are considered to be two key drivers of NECs, but in
NETs these mutations are rarely observed (104‑107). Double
inactivation of the TP53 and RB1 genes is one of the genetic
signatures of SCLC (108). The presence of these inactivated
mutations is used as a predictive marker of the response to
platinum‑based CHT in lung and GEP‑NECs (104,105,108). In
addition, the loss of RB1 is associated with superior responses
to platinum salts in both lung and pancreas NECs (104,109),
whilst the presence of p53 staining in colorectal NECs is
associated with weaker responses to platinum‑based CHT and
worse prognosis (105). In GEP‑NECs, p53 IHC is currently
under examination as a possible diagnostic, prognostic and
predictive method for GEP‑NECs, which was reported in
several studies with frequencies of p53 immunoreactive cells
ranging from 20‑100% (103), where mutations in TP53 associ‑
ates poor survival, moreover TP53 being the most prevalent
mutation in NECs (105,110).
Expression markers
Insulinoma‑associated protein 1 (INSM1). INSM1 is a
zinc‑nger family of transcription factors that serves a role
in neurogenesis and neuroendocrine cell differentiation (111).
CIOBANU et al: CURRENT AND POTENTIAL MARKERS IN NEUROENDOCRINE NEOPLASMS
6
INSM1 confers certain advantages in NECs as it more sensitive
compared with traditional general neuroendocrine markers,
such as CgA or synaptophysin, since the clear nuclear expres‑
sion pattern of INSM1 facilitates its accurate interpretation
and quantication whilst reducing the incidence of staining
artifacts (65).
The Notch/hairy and enhancer of split‑1 (Hes‑1) signaling
pathway serves a key role in tumor growth and development,
which has also been reported to inhibit INSM1 (112). INSM1
has recently been reported to be an important biomarker in
the diagnosis of SCLC as it serves as an important factor in
ASCL1‑driven pathways. ASCL1 is needed for the protein
expression of NE molecules and in the development of lung
NE cells (113,114). Furthermore, functional interactions
between ASCL1 and the Notch1‑Hes1 pathway have been
reported (113). As aforementioned, the Notch1‑Hes1 pathway
is involved in the suppression of INSM1 expression (112).
Moreover, the implication of INSM1 suppression is being
studied as a modulator factor in PanNETs, which could be
connected to the Notch1‑Hes1 signaling pathway (112,115).
Tanigawa et al (112) revealed that INSM1 expression was
positive in all cases of PanNETs but negative in cases of pancre‑
atic ductal adenocarcinoma (PDAC), rendering it a potential
marker for distinguishing between PanNETs and PDAC. In
addition, INSM1 has also been found to be a potential marker
for SCLC, since a number of studies reported that INSM1
expression was present in 97‑100% SCLC cell lines, which
appeared to be more sensitive and specic compared with CgA
and l‑3,4‑dihydroxyphenylalanine decarboxylase (111,113).
Furthermore, recent studies reported the superior sensitivity of
INSM1 for the diagnosis of NENs regarding their neuroendo‑
crine origin (65,116). Bellizzi et al (65) studied a cohort of 93
NECs, where 95% sensitivity was found for INSM1 compared
with 83 and 82% for CgA and synaptophysin, respectively.
NETest‑a transcriptomic signature of NETs. The NETest uses
multianalyte assays with algorithmic analyses (MAAAs),
which is a novel method including procedures that incorpo‑
rate results derived from multiple assays and can increase
both the sensitivity and specicity (117). This assay involves
mRNA isolation, cDNA synthesis followed by the subse‑
quent quantitative‑PCR measurement of 51 circulating NET
marker genes (117). These genes were chosen based on the
analyzed microarray datasets containing the cellular proles
of fresh frozen tumor samples and whole blood samples from
patients diagnosed with NET to characterize the expression
patterns (117). Finally, a multi‑analyte liquid biopsy represen‑
tative for NETs was used to determine the biological activity
of the tumor and therefore clinical status of the patient (118).
Results are presented as the numeric score (NET score) that
include the following three categories: i) Low, ≤21‑40%;
ii) intermediate, 41‑79%; and iii) high (biologically aggres‑
sive), ≥80%. The PCR test is standardized and reproductible;
moreover, diet, proton pump inhibitors medication, age,
sex and ethnicity do not interfere with the accuracy of the
test (119,120). In addition, MAAA is reproducible at multiple
time points, which provides real‑time NET scores (118,120).
In a cohort of 100 patients with three different types of
tumors (GEP, lung bronchopulmonary NETs and unknown
origin tumors), the NETest test had a reported diagnostic
sensitivity of 96%, which was >90% effective in combination
with diagnostic imaging for guiding treatment decisions (121).
This previous study also demonstrated that a low NET score
(≤40%) was associated with superior outcomes, where the
PFS was not reached, whilst intermediate to high NETest
scores (41‑100%) were associated with signicantly shorter
PFS and treatment failures (121). In addition, the NETest has
been documented to be a useful tool for the detection of lung,
thymic, pancreatic and gastrointestinal tract NETs, as well as
paragangliomas and pheochromocytomas with ≥90% accu‑
racy (39). Moreover, the NETest can predict aggressive tumor
behavior in other NENs (122,123), or the outcome following
tumor resection and efcacy of medical treatments, such as
somatostatin analogues (SSAs) or peptide receptor radio‑
nuclide therapy (PRRT) (39).
A recent meta‑analysis was conducted to assess the
eligibility of NETest as a biomarker tool in the field of
oncology (124). The results supported the utility of this test
as a diagnostic tool for GEP and bronchopulmonary NETs,
which reported an accuracy of 85% in differentiating between
stable and progressive disease whereas a specicity of ~90%
was reported (124).
Aristaless related homeobox (ARX)/pancreatic and duodenal
homeobox 1 (PDX1) expression. ARX and PDX1 are regula‑
tory proteins involved as epigenetic modiers in pancreatic
development (86). The assessment of ARX/PDX1 expression
has been previously studied as a potential pre‑operative risk
stratication marker for PanNETs (125). Since >50% PanNETs
already have liver metastases at rst presentation, novel strate‑
gies are emerging for the other 50% with aims to reduce the
risk of metastasis (126). Although the choice of surgery for
the primary tumor can reduce the risk of metastasis, it is also
associated with increased risk of post‑surgery morbidity and
mortality. Therefore, apart from tumor size, transcription
factors ARX and PDX1 alongside ATRX/DAXX mutations
and the status of ALT were studied as prognostic markers
in resected NF PanNETs (96). Cytological specimens of
PanNETs obtained by endoscopic FNA was previously studied
using the IHC staining of ARX, PDX1 and telomere‑specic
uorescence in situ hybridization to detect ALT (125). Positive
ALT activity and ARX expression in the tumor coupled with
negative PDX1 staining was documented to predict metastatic
phenotype for the stratication of patients into the low‑ or
high‑risk groups preoperatively (125).
MicroRNA (miRNA or miR) markers. miRNAs are a class of
RNAs that do not encode protein and are ~22 nucleotides in
length (127). They typically regulate post‑transcriptional gene
expression by targeting mRNA molecules (127). Circulating
miRNAs can be used as minimally invasive biomarkers of
ovarian, cervical or breast cancer as they are readily detectable
in a wide variety of biouids, including plasma, serum and
saliva (128).
The global microRNA expression patterns were studied
in normal pancreas, PanNETs and acinar carcinomas in order
to assess the role of microRNAs in malignant transforma‑
tion and progression (129). miR‑103 and miR‑107 expression
levels were found to be higher whereas the level miR‑155
expression was lower in PanNETs compared with the normal
EXPERIMENTAL AND THERAPEUTIC MEDICINE 22: 1479, 2021 7
pancreas (129). Furthermore, miR‑21 expression level appears
to be higher in the plasma of patients with PanNETs compared
with that in patients with chronic pancreatitis (129). Therefore,
it can be applied both as a diagnostic and a prognostic tool for
PanNETs, since higher levels of miR‑21 expression and high
Ki‑67 proliferation index were associated with the presence of
metastases (129,130).
It was recently reported that in tumors without a known
primary site, miRNA expression can be used to facilitate
diagnosis (131). miRNA expression proles were previously
analyzed in four pathological types of GEP‑NETs, including
samples from pancreatic, ileal, appendiceal and rectal
NETs (131). The results were promising, as the midgut
NETs (ileum and appendix) could be discriminated from
non‑midgut NETs (rectum, pancreas) according to miR‑615
and miR‑92b expression (131). In addition, ileal NETs could be
discriminated from appendiceal NETs according to miR‑125b,
miR‑192 and miR‑149 expression, whilst rectal NETs could be
distinguished from pancreatic NETs based on miR‑429 and
miR‑487b expression (131).
Due to their stability in the circulation and abundance,
cell‑type and disease stage specificity and their reported
roles in a number of biological processes, miRNAs have been
investigated in various studies. However, due to the lack of
consistency in the reported signatures (between results that
used tissue and those that used circulating blood) for the same
disease and the lack of standardization methods with accurate
techniques, further research is required (132,133).
Methylation markers. Epigenetic events occur depending on
the subtype of NETs (134). DNA hypermethylation is an early
event that frequently occurs during cancer initiation and can
dictate the rate of disease development and progression (135).
The ‘Hypermethylation phenotype’ was associated with poorer
OS and with more progressive disease in PanNETs (135).
O‑6‑methylguanine‑DNA methyltransferase (MGMT) for
NETs but not for NECs. The role of the MGMT enzyme is
to repair DNA lesions as a result of alkylating agents usually
used in NENs. Loss of MGMT function occurs as a result of
epigenetic events, such as hypermethylation, of the MGMT
gene promoter (136). This in turn leads to the loss of MGMT
protein expression, which can be detected using IHC or
detected on the gene level by methylation analysis (for example
using methyl‑specic PCR or pyrosequencing). Analysis of
the MGMT status can be used to predict the prognosis and
response to alkylating agents that induce DNA damage in
well‑differentiated NENs (107). Previous studies showed
that reduced MGMT expression is associated with increased
rates of treatment response to temozolomide, dacarbazine and
streptozotocin CHT in digestive and lung NENs (136,137).
Circulating biomarkers
Cell‑free DNA (cfDNA). The impor tance of tumor‑specic alter‑
ations in cell‑free DNA (cfDNA) in liquid biopsies is becoming
increasingly recognized, which can either complement or
replace tissue biopsies for several types of cancer, including
NSCLC with mutations in the EGFR gene (138,139). cfDNA
consists of a proportion of circulating tumor DNA (ctDNA) in
the blood plasma, which originates from the tumor following
apoptosis, necrosis and active secretion (140,141). cfDNA can
be used as a biomarker for cancer, as patients with patients are
reported to have greater levels of plasma cfDNA compared with
tumor‑free controls (142); in addition, high levels of cfDNA are
also described in other diseases, such as autoimmune disor‑
ders (143). However, further studies in NENs are required to
verify the utility of cfDNA as a proling tool. Boons et al (141)
rst reported that the presence of ctDNA through the identi‑
cation of copy number variations and tumor specic point
mutations using shallow whole genome sequencing and
droplet digital PCR, respectively can be used to differentiate
between metastatic and localized PanNET. The results of
this study demonstrated that ctDNA is found in the plasma
samples of patients with metastatic disease. This was shown
by tumor‑specic variants that were obtained through whole
exome sequencing (WES) analysis of primary tumor tissue and
germline DNA, in comparison with localized PanNETs where
when genotyping variants in cfDNA, the variants could not
be detected. Therefore, cfDNA is a candidate as an alternative
biomarker to tissue biopsies for molecular proling.
Circulating tumor cells (CTCs). CTCs are typically released
into the blood of patients who have undergone epithe‑
lial and mesenchymal transition, which cause metastatic
disease (13,144). The presence of CTCs at the moment of
recruitment/initial evaluation in the blood samples of patients
with midgut, pancreas, bronchopulmonary and of unknown
primary metastatic NENs was previously associated with
worse PFS and OS (145). In addition, CTCs are associated
with higher tumor grades and burden, high levels of CgA and
higher Ki‑67 indices in G1 and G2 midgut and pancreatic
NETs (146). Their potential predictive role was previously
studied in a cohort of 138 patients with metastatic NENs,
where 41 (29.7%) received long‑term SSAs. Changes in repeat
CTC count at 3‑5 weeks after initiation of therapy were asso‑
ciated with both progressive disease and OS (147). Improved
survival was recorded in patients who did not have CTCs at
baseline and after therapy in addition to those who presented
with >50% CTC reduction after treatment (147). However,
CTC lacks sensitivity and specicity as a diagnostic tool for
different types of NENs (13). This limitation is currently under
investigation in a multicenter, exploratory CalmNET phase IV
study (ClinicalTrials.gov Identier, NCT02075606), which is
monitoring a relatively homogeneous group of patients with
F G1‑G2 midgut NETs treated with lanreotide autogel. This
study is attempting to evaluate the predictive power of CTC
count on clinical outcome, PFS and quality of life (148).
Molecular imaging as biomarkers
Somatostatin receptors agonists. Expression of SSTR in the
majority of NENs, particularly subtype 2, can be imaged by
labelling SSAs with a radionuclide, which is typically 68Ga,
using a chelate, such as 1,4,7,10‑tetraazacyclododecane‑1,4,7,1
0‑tetraacetic acid (DOTA) (149,150).
At present, two types of molecular imaging that can be
used to target SSTR in clinical practice: 111In‑pentetreotide
(OctreoScan) and 68Ga‑DOTA‑Phe1‑Ty r 3‑ O ctreot ide
(TOC), 68Ga‑DOTA‑NaI3‑Octreotide (NOC) or
68Ga‑DOTA‑Tyr3‑Octreotate (TATE). 68Ga‑DOTA‑
TATE/TOC/NOC‑PET/CT confers higher scanner
CIOBANU et al: CURRENT AND POTENTIAL MARKERS IN NEUROENDOCRINE NEOPLASMS
8
sensitivity, superior spatial resolution and require lower radia‑
tion doses (151). Although all three radiotracers (DOTA‑TOC,
DOTA‑NOC and DOTA‑TATE) have good afnity for SSTR
types 2 and 5, 68Ga‑DOTA‑NOC exhibits higher afnity for
SSTR type 3 (152). However, 68Ga‑DOTA‑TATE confers
superior diagnostic precision for the nuclear imaging of NENs
and is currently used in USA, whilst 68Ga‑DOTA‑TOC‑PET is
mainly used clinically in the European Union (153,154).
68Ga‑DOTA‑TATE‑PET has previously demonstrated
its superiority over Octreoscan for the location of primary
GEP‑NETs, with a 95.1% detection rate compared with
30.9 and 45.5% from other conventional imaging modali‑
ties, respectively (155). This outcome changed the medical
recommendation in 32.8% of the patients (155). Results from
68Ga‑DOTA‑TOC‑PET/CT imaging also demonstrated a high
afnity to SSTR2 expression derived from IHC and can serve
as a predictive marker for patient response to treatment with
PRRT (156,157). In addition, 68Ga‑DOTA‑TOC‑PET/CT can be
used to select patients who may potentially benet from SSAs
and PRRT, as a high tumor uptake of 68Ga‑DOTA‑TOC‑PET/CT
can be useful in the treatment selection of the patients (158).
PRRT is a key second‑line treatment option for G1 or G2
midgut NETs with disease progression on SSA treatment (91).
PRRT can be used to identify radiation delivered by radionu‑
clides, such as lutetium‑177 (177Lu) or yttrium‑90 (90Y), to NET
cells following internalization after binding to SSTR (151).
However, the expression of SSTRs is currently being
studied as a predictive marker for treatment response (13).
In the NETTER‑1 prospective randomized phase 3
clinical trial (ClinicalTrials.gov Identifier, NCT01578239),
177Lu‑DOTA‑TATE demonstrated its superiority compared
with high‑dose octreotide i n prolonging the PFS in patients with
midgut NETs (PFS at month 20, 65.3% for 177Lu‑DOTATATE;
10.8% for high‑dose octreotide group) (159).
Somatostatin receptors antagonists. Higher tumor uptake of
radiolabeled somatostatin receptor antagonists their poten‑
tial role has been studied for diagnostic and therapeutic
approach in NETs (160). Previous studies reported higher
sensitivity and diagnostic accuracy with increased image
contrast for 68Ga‑NODAGA‑JR11 (68Ga‑OPS202), a SSTR‑2
antagonist, compared with agonists 68Ga‑DOTA‑TATE
and 68Ga‑DOTA‑TOC, for staging G1 and G2 GEP‑NETs
(ClinicalTrials.gov identier, NCT02162446) (160,161). A
‘theragnostic pair’‑68Ga/177 Lu‑DOTA‑JR11 combination was
also investigated in a single‑center study (ClinicalTrials.gov
identier, NCT02609737). Although there are indications
that it binds to more cell types compared with DOTA‑TATE
or DOTA‑TOC in low‑grade NETs, this investigation
remain in progress at present. Similarly, a peptide ligand,
68Ga‑DOTA‑bombesin, which can bind to the gastrin
realizing peptide receptor in prostate cancer cells (162),
is another example of this receptor system that is under
evaluation for NENs. Its rst appl ication for PET imaging in
humans for prostate and breast cancer has been previously
reported (162).
18F‑uoro‑deoxyglucose (FDG)‑PET/CT (18F‑FDG‑PET/CT).
18F‑FDG‑PET/CT avidity, especially for detecting G3 NET, was
reported to be an indirect marker of proliferative activity in the
tumor, showing a higher sensitivity (87.5%) than somatostatin
receptor scintigraphy (87.6%) for detecting rapidly progressive
disease (163). In a retrospective study, 18F‑FDG avidity was
measured quantitatively as a potential prognostic marker in a
cohort of 89 patients with metastatic GEP‑NETs (164). These
patients were divided into three groups based on the ratio of
standardized uptake value (SUV) max of the lesion that had
the highest FDG uptake as compared to normal liver uptake of
FDG (tumor‑to‑liver T/L SUV ratio) (ratio ≤1, 1‑2.3 and >2.3).
These three categories associated positively with OS (median
OS not reached after 114 months for patients with T/L SUV
ratio ≤1 vs. 55 months for patients with T/L SUV ratio of 1‑2.3
vs. 13 months for patients with T/L SUV ratio >2.3) (164).
NET‑PET score. Somatostatin receptor imaging (SRI)
is currently considered the gold standard for detecting
well‑differentiated NETs. However, it has a number of limita‑
tions for detecting high grade NETs due to the possibility of
false‑negatives, since these types of tumors do not express
SSTRs (156). Although 18F‑FDG‑PET can be used for staging
G3 NECs, it is more suited for predicting the prognosis of
well‑differentiated NETs, where higher levels of uptake were
associated with an increased risk of early progression (163).
By contrast, lower levels of uptake is associated with a less
aggressive phenotype of the tumor (163). Chan et al (165)
therefore proposed a grading system combining these two
nuclear imaging techniques (SRI and 18F‑FDG‑PET) as a
single parameter, named ‘NET‑PET’, which was found to
associate with OS. This scoring system is designated into ve
risk category groups: i) Grade P0, negative uptake for both
scans; ii) grade P1, purely STTR‑positive lesions without
FDG uptake above background; iii) grades P2‑P4, interme‑
diate categories; and iv) grade P5, presence of signicant
FDG‑positive/STTR‑negative disease (166). The NET‑PET
score may influence the initial management method of
patients with well‑differentiated metastatic midgut NET,
since the ENETS guideline (167) recommends CHT or SSA
as the rst‑choice treatment option. During the initial phases
of SSA therapy, there is no consensus on the upper cut‑off
value of the Ki‑67 proliferation index (165). In this case, the
NET‑PET score would favor SSA treatment if there is high
SRI uptake and low FDG uptake, whilst the contrary would
favor CHT (168). The same line of reasoning can be made
during patient selection for PRRT, where high SRI uptake but
low FDG uptake would suggest PRRT as a viable treatment
option, whilst higher FDG uptake and low SRI uptake would
indicate likelihood of PPRT resistance (169). Since this was
a retrospective analysis, it remains to be elucidated how the
individual intermediate classications can inuence the prog‑
nosis and treatment decision in a prospective study.
Tumour growth rate as an early biological marker.
Accumulating data suggest that the response evaluation
criteria in solid tumors (RECIST 1.1) has several limitations
in predicting the response to different types of systemic treat‑
ments (ST) (34,170). According to RECIST 1.1, the majority
of patients with G1‑2 GEP‑NETs would be classied as having
‘stable disease’ (170). However, even non‑responders can
survive for a long period without disease progression, since it
is known that well‑differentiated GEP‑NETs have a relatively
EXPERIMENTAL AND THERAPEUTIC MEDICINE 22: 1479, 2021 9
slow growth rate (34). Therefore, is important to discover
a tumor marker that can identify patients who are at high
risk of disease progression at the early stage (171). Previous
studies reported TGR to be a dynamic marker, which analyzes
images from two examinations and the time between the
examinations and can reveal benecial quantitative informa‑
tion regarding the percentage of change in the tumor volume
each month (171,172). The GREPONET I study conrmed
that the TGR measured at 3 months (TGR3m) after starting ST
or watch and wait (WW) treatment with a cut‑off of 0.8%/m
(m, percentage of the change of the tumor size in one month),
could be used in clinical practice to monitor the treatment
response in NETs for the early prediction of PFS (34). In addi‑
tion, an increased TGR3m (≥0.8%/m) was found to associated
with shorter PFS, whilst a decreased TGR3m (<0.8%/m‑) was
found to associate with longer PFS (34). This nding suggests
that patients with high TGR3m should be followed up more regu‑
larly, whilst those with lower TGR3m can receive imaging less
regularly to avoid unnecessary radiation (34). Subsequently,
the GREPONET II study explored whether beginning any ST
including WW can induce any changes in TGR, which was
dened as TGR3m‑TGR0 (TGR0 was calculated by comparing
the baseline and imaging examination performed within
1 year before the baseline scan) (35). Since it was expected
that TGR can be used for monitoring treatment change with no
impact on PFS, further study is required.
5. Conclusions
Selection of the optimal treatment option for patients with
NEN is difficult due to the heterogeneity in the tumor
physiology and varying degrees of aggressiveness. There is a
demand for multidisciplinary tumor management guidelines
driven by data derived from modern radiology and molecular
proling techniques, to inform the optimal medical decision.
Overall, further studies integrating a combination of markers
based on tumor genomics and a large spectrum of radiological
techniques, such as molecular imaging, would be better placed
for shaping the future of clinical NEN research. The present
review highlights the importance of a second opinion for
improving the method of prognostic stratication and choice
of personalized treatment strategies.
Acknowledgements
Not applicable.
Funding
No funding was received.
Availability of data and materials
Not applicable.
Authors' contributions
OAC wrote the literature review. SF and SM critically
reviewed the manuscript. Data authentication is not applicable.
All authors read and approved the nal manuscript.
Ethics approval and consent to participate
Not applicable.
Patient consent for publication
Not applicable.
Competing interests
The authors declare that they have no competing interests.
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