Review Article

Algorithmic Approach to Neuroendocrine Tumors in Targeted

Biopsies: Practical Applications of Immunohistochemical Markers
Kai Duan, MD1,2 and Ozgur Mete, MD, FRCPC1,2,3

Neuroendocrine tumors (NETs) constitute a heterogeneous group of neoplasms with distinct biological behaviors, depending on the site
of origin and the degree of tumor proliferation. Although advances in biochemical and radiological modalities have enhanced the ability to
detect NETs, tissue diagnosis remains the gold standard to assess tumor characteristics for treatment decision making. In an era with
growing demands for precision diagnostics based on smaller tissue samples, immunohistochemistry has become an indispensable tool in
the pathologist’s repertoire. In conjunction with clinical findings and cytomorphology, complementary use of 1) markers of neuroendocrine
differentiation, 2) markers confirming epithelial nature, 3) markers of cellular proliferation, 4) transcription factors and hormonal markers,
as well as 5) predictive and prognostic markers may be necessary to guide patient management in NETs. The current review summarizes
common applications of these immunohistochemical markers when confronted with a potential neuroendocrine neoplasm, and proposes a
stepwise algorithmic approach to avoid diagnostic errors in targeted biopsies. Cancer Cytopathol 2016;124:871-84. V
C 2016 American Can-

cer Society.

KEY WORDS: biomarkers; hormones; immunohistochemistry; neuroendocrine tumor; paraganglioma; poorly differentiated neuroendocrine
carcinoma; transcription factors.

INTRODUCTION
Neuroendocrine tumors (NETs) encompass a wide spectrum of neoplasms arising from various anatomic locations, with diverse
biological behaviors.1–6 Despite advances in diagnostic imaging and biochemical techniques, some patients still present with
unknown primary site prior to the pathological examination.1–3,7–9 Accurate detection and subtyping are critical for patient man-
agement and can be challenging on small biopsies.5,10,11 In particular, assessment of tumor proliferation and confirmation of the
primary site are frequently requested, given their treatment implications.1–3,7,8 In an era with increasing demands for precise
diagnosis based on smaller tissue samples, immunohistochemistry has become an indispensable adjunct tool to accurately classify
these tumors.2,4,7–9 The current review summarizes common applications of biomarkers through an algorithmic approach to
diagnose NETs in targeted biopsies (Table 1).

Confirmation of Neuroendocrine Differentiation

The diagnosis of a suspected neuroendocrine neoplasm typically begins by confirming neuroendocrine differentiation, using a
panel of general neuroendocrine immunohistochemical markers.1,7,8 In the setting of a metastatic tumor of unknown primary,
this may be part of a wider immunohistochemical panel to identify the site of origin.2,12,13 Although several biomarkers (includ-
ing CD56, CD57, protein gene product 9.5 [PGP9.5], and neuron-specific enolase) have been described to date, the most
widely used and reliable neuroendocrine markers are chromogranin A and synaptophysin (Figs. 1 and 2).1,2,14–16 While chromo-
granin A is the most specific marker, synaptophysin is often regarded as the most sensitive marker in the diagnosis of
Corresponding author: Ozgur Mete, MD, FRCPC, Department of Pathology, University Health Network, 200 Elizabeth St, 11th Fl, Toronto, ON M5G 2C4, Canada; Fax:
(416) 340-5517; ozgur.mete2@uhn.ca

We thank Dr. Gilda Santos (Department of Laboratory Medicine Program, University Health Network) for providing some of the photomicrographs used in Figures 1
and 2.
1
Department of Pathology, University Health Network, Toronto, Ontario, Canada; 2Department of Laboratory Medicine and Pathobiology, University of Toronto, Toronto,
Ontario, Canada; 3Endocrine Oncology Site Group, Princess Margaret Cancer Centre, Toronto, Ontario, Canada

Received: June 19, 2016; Accepted: June 27, 2016

Published online August 16, 2016 in Wiley Online Library (wileyonlinelibrary.com)

DOI: 10.1002/cncy.21765, wileyonlinelibrary.com

Cancer Cytopathology December 2016 871

Review Article
TABLE 1. Overview of Immunohistochemical Markers
for NETs
1) Markers for neuroendocrine differentiation
Chromogranin A (most specific; recommended)
Synaptophysin (most sensitive; recommended)
CD56, CD57, PGP9.5, and neuron-specific enolase (less specific markers,
not recommended for well-differentiated NETs)
2) Markers for epithelial differentiation
Cytokeratins (AE1/AE3 and CAM5.2)
3) Marker for tumor proliferation
Ki-67/MIB-1
4) Markers for site of origin
Pituitary: Pit-1, T-pit, and SF-1
Parathyroid: PTH, GCM-2, and GATA-3
Medullary thyroid carcinoma: TTF-1, monoclonal CEA (diffuse), and calcito-
nin/calcitonin-gene related peptide
Pulmonary: TTF-1, calcitonin/calcitonin-gene related peptide, bombesin, and
serotonin
Midgut: CDX-2 (strong and diffuse) and serotonin
Pancreatic: ISL-1, PDX-1, and CDX-2 (6)a
Duodenal: ISL-1, PDX-1, xenin, and CDX-2 (6)a
Hindgut/rectal: PAP, pancreatic polypeptide, glucagon-like peptide 1, peptide
YY, ISL-1 (6), CDX-2 (6),a and serotonin (6)
Paraganglioma: tyrosine hydroxylase and GATA-3
5) Markers with potential prognostic or therapeutic implications
SSTRs 1-5
Hormones
E-cadherin and b-catenin
Pancreatic NETs: DAXX, ATRX, menin, CEACAM1, CK19, CD117, PTEN, PR,
cyclooxygenase-2, p21, p18, and Rb
SDHB immunohistochemistry
Mismatch repair proteins (MLH1, MSH2, MSH6, and PMS2)
Abbreviations: 6 , variable immunohistochemical expression; ATRX, ATP-
dependent helicase ATRX, X-linked helicase II; CDX-2, caudal type homeobox
2; CEA, carcinoembryonic antigen; CEACAM1, carcinoembryonic antigen-
related cell adhesion molecule 1; CK19, cytokeratin 19; DAXX, death domain-
associated protein; GATA-3, GATA-binding protein 3; GCM-2, glial cell missing 2
protein; ISL-1, insulin gene enhancer protein; MLH1, MutL homolog 1; MSH2,
MutS protein homolog 2; MSH6, MutS protein homolog 6; NET, neuroendocrine
tumor; PAP, prostatic acid phosphatase; PDX-1, pancreatic and duodenal
homeobox 1; PGP.9.5, protein gene product 9.5; Pit-1, pituitary-specific
transcription factor 1; PMS2, PMS1 homolog 2; PR, progesterone receptor;
PTEN, phosphatase and tensin homolog; PTH, parathyroid hormone; SDHB,
succinate dehydrogenase B; SF-1, Steroidogenic factor 1; SSTRs, somatostatin
receptors; TTF-1, thyroid transcription factor-1.
a
Although well-differentiated neuroendocrine tumors (WDNETs) from the pan-
creas, foregut, and hindgut can sometimes express CDX-2 positivity, the stain-
ing pattern is usually weak and patchy compared with the strong and diffuse
staining noted in midgut enterochromaffin cell WDNETs.
well-differentiated NETs.17,18 It should be noted that the level
of chromogranin A expression may vary according to the
degree of tumor differentiation (Fig. 2).2 Thus, focal, weak, or
even absent chromogranin A staining may be noted in poorly
differentiated neuroendocrine carcinomas (NECs) (Fig. 2);
however, synaptophysin should still be present in these cases.
Nonetheless, it is important to note that positivity for synapto-
physin alone does not warrant a diagnosis of NET in all cases.
For example, solid pseudopapillary tumor of the pancreas and
adrenal cortical carcinoma can also express synaptophy-
sin.1,19,20 On another note, some NETs, including the major-
ity of L-cell NETs of hindgut origin, may preferentially
express chromogranin B instead of chromogranin A.1,21 This
872
issue is of clinical significance as most laboratories do not have
access to chromogranin B immunohistochemistry. In light of
these findings, the concurrent use of chromogranin A and syn-
aptophysin is recommended to accurately confirm or exclude
neuroendocrine differentiation in a suspected well-
differentiated NET. In addition, it should be noted that some
NETs can be seen in the context of mixed tumors (ie, mixed
adeno-NEC), whereas other tumors may demonstrate neuro-
endocrine differentiation (ie, adenocarcinoma with focal diver-
gent neuroendocrine differentiation); the distinction of these
entities can be difficult in small biopsy samples.1,22–24 How-
ever, the extent of neuroendocrine marker staining in adeno-
carcinoma with divergent neuroendocrine differentiation is
generally <30% of the tumor volume.25,26 Therefore, quanti-
fication of neuroendocrine marker staining is pertinent,
although it may not reflect the characteristics of the overall
tumor in a cytology specimen. Accurate recognition of these
diagnostic pitfalls, along with strict correlation of cytomor-
phology and immunohistochemical findings, is crucial to pre-
vent misdiagnosis.
Confirmation of Epithelial Nature
After confirmation of neuroendocrine differentiation, a panel
of general epithelial biomarkers (cytokeratins AE1/AE3,
CAM5.2, and/or CK18) is recommended to exclude a poten-
tial paraganglioma (Figs. 1 and 3).1,8,16,27 This distinction is
crucial as the management of paraganglioma can differ from
that of other NETs.1,10,16,27,28 Although a small percentage of
NETs may stain negative for epithelial markers, a paragan-
glioma should always be excluded in a cytokeratin-negative
NET.1,16 Paragangliomas (including pheochromocytomas,
which are intra-adrenal sympathetic paragangliomas) are
“specialized” neuroendocrine neoplasms arising from neural
crest-driven endocrine organs (known as paraganglia) that pro-
duce catecholamines (epinephrine, norepinephrine, and dopa-
mine).1,27,28 Recent evidence suggests that up to 40% of these
neoplasms are associated with genetic susceptibility, with clini-
cal implications for genetic testing.16,27,29–34 Although lack of
immunohistochemical expression of transcription factors (ie,
thyroid transcription factor 1 [TTF-1], caudal type homeobox
2 [CDX-2], paired box 8 [PAX-8], islet-1, pancreatic and
duodenal homeobox 1 [PDX-1]) supports a diagnosis of a
paraganglioma in a cytokeratin-negative NET, this finding
should be corroborated with positivity for tyrosine hydroxy-
lase, which is the rate-limiting enzyme in catecholamine syn-
thesis (Fig. 3).1,27,29,30 Demonstration of S100-positive
sustentacular network also favors a diagnosis of paraganglioma
Cancer Cytopathology December 2016
 Algorithmic Approach to NETs in Targeted Biopsies/Duan and Mete
Figure 1. Well-differentiated neuroendocrine tumor (A: Giemsa stain) with (B) chromogranin A, (C) synaptophysin, and (D) AE1/AE3
positivity.
in surgical specimens; however, S100-positive sustentacular
cells can be identified in other NETs, including pituitary
adenomas, gastroenteropancreatic and pulmonary
1,16,35,36
NETs. Therefore, the identification of sustentacular
cells is not helpful in this distinction. The real challenge arises
when confronted with a cytokeratin-negative neuroendocrine
neoplasm that is also negative for tyrosine hydroxylase. This
can occur in a subset of nonfunctioning paragangliomas which
are mostly segregated in the head and neck region.1,16,30
Although some head and neck paragangliomas can still dem-
onstrate positivity for tyrosine hydroxylase, the majority will
have focal-to-weak or absent staining for tyrosine hydroxy-
lase.1,16,30 In this specific context (keratin-negative and
tyrosine hydroxylase-negative NET), positivity for trans-acting
T-cell-specific transcription factor (GATA-binding protein 3
[GATA-3]) may help to support a diagnosis of paraganglioma
(Fig. 3); however, it should be noted that GATA-3 is also
expressed in nonendocrine tumors as well as parathyroid neo-
plasms (which are positive for neuroendocrine
markers).1,16,30,37–41 Ultimately, absent staining for relevant
transcription factors (ie, TTF-1, CDX-2, glial cell missing 2
Cancer Cytopathology December 2016
protein [GCM-2], etc), hormones (ie, parathyroid hormone
[PTH] and monoclonal calcitonin) and monoclonal carcino-
embryonic antigen (CEA) may also be required to render a
diagnosis of GATA-3-positive and tyrosine hydroxylase-
negative paraganglioma in the head and neck region.1,16
Exceptional examples of paragangliomas, namely cauda equina
paragangliomas and gangliocytic paragangliomas (comprised
of a mixture of epithelioid neuroendocrine cells, Schwann-like
cells, and scattered ganglion-like cells) may also demonstrate
positivity for cytokeratin, a phenomenon believed to be related
to divergent epithelial differentiation.1,42
Assessment of Tumor Proliferation
After confirmation of neuroendocrine and epithelial differen-
tiation, the next step pertains to differentiating a well-
differentiated NET (WDNET) from a poorly differentiated
NEC.1,7,8,10 While tumor cytomorphology, including
mitotic activity, may be sufficient in making this distinction,
the concurrent use of a proliferative biomarker (Ki-67/MIB-
1) is generally advised.1,5,7,8,43–46 The classification of NET
is site- and grade-dependent, requiring careful evaluation of
873
Review Article

Figure 2. Poorly differentiated neuroendocrine carcinoma (NEC) of small cell type. In this composite figure, pitfalls associated with
poorly differentiated NECs are illustrated. The fine-needle aspiration biopsy identified a small cell NEC (A: Papanicolaou stain). (B)
Unlike well-differentiated neuroendocrine tumors, positivity for chromogranin A can be weak or even absent. Dot-like paranuclear
chromogranin-A staining can be identified in some cases. Positivity for synaptophysin and (C) CD56 often remain in poorly differenti-
ated NECs. (D) High proliferative activity is often reflected in high Ki-67 labeling.

cytomorphology along with proliferative features of the
tumor.1,5,7,8,43–46 Although the most recent World Health
Organization classification still does not recognize the impact
of the Ki-67 index in the grading of mediastinal NETs, the
distinction between a poorly differentiated NEC (ie, small
cell NEC, large cell NEC) and a WDNET is usually not a
major challenge in cytology specimens.1,5,7,8,43–46 Nonethe-
less, the use of Ki-67 may still be helpful (Fig. 2).45 The real
challenge arises when distinguishing between low-grade ver-
sus intermediate-grade NETs. The proliferation index in
fine-needle aspiration cell blocks should be interpreted with
caution.43,44,47–52 Because neuroendocrine neoplasms can be
quite heterogeneous with respect to their proliferative activ-
ity, small biopsy specimens often fail to meet the minimum
requirements for formal grading.43,44,47–52 The reported
concordance between endoscopic ultrasound-guided fine-
needle aspiration cytology versus surgical pathology in NET
grading (using the Ki-67 index) varies from 50% to
89.5%.43,44,47–52
Determination of the Site of Origin
Given the treatment implications, a diagnosis of well-
differentiated NET may require further immunohistochemical
workup to establish the site of origin, particularly if this infor-
mation is ambiguous to the treating clinician.1,7,8,53 In con-
trast, determination of the primary site in poorly
differentiated NEC is often not requested, given the uniform-
ity in treatment modalities and disease behavior.1,5,7,10,54 In
combination with clinical and cytomorphological findings, a
panel of markers for transcription factors and hormones offers
the highest sensitivity and specificity to identify the site of ori-
gin.1,2,7,8,17 A detailed description of each marker is beyond
the scope of this paper, and has been reported elsewhere.1,8
For brevity, we will review common applications of immuno-
histochemistry, pertaining to frequent sites where NETs can
arise (head and neck, thorax, pancreas, and various parts of the
gastrointestinal tract). In the setting of a metastatic WDNET
of unknown primary, we recommend a panel approach using
multiple immunohistochemical markers simultaneously.1,8,55

874 Cancer Cytopathology December 2016

 Algorithmic Approach to NETs in Targeted Biopsies/Duan and Mete

Figure 3. Keratin-negative neuroendocrine tumor should alert the diagnostician to the possibility of a paraganglioma. A young patient
presented with an enlarged neck mass. The fine-needle aspiration and synchronous core needle biopsy specimens demonstrated a
neuroendocrine neoplasm (A: core needle biopsy; H & E). Immunohistochemically, the tumor cells were positive for (B) chromogranin
A and (C) synaptophysin, but were negative for (D) AE1/AE3 and CAM5.2 (not shown). The initial panel raised the possibility of a par-
aganglioma; (E) positivity for tyrosine hydroxylase confirmed the diagnosis of paraganglioma. (F) GATA-binding protein 3 (GATA-3)
was also expressed in the tumor cells. There was no loss of succinate dehydrogenase B (SDHB) expression to suggest succinate dehy-
drogenase (SDHx)-related pathogenesis (not shown).

Extra-abdominal locations calcitonin gene-related peptide), as well as other hormones

As a general rule, positivity for TTF-1 in a WDNET favors a
primary site from either the head and neck (specifically medul-
lary thyroid carcinoma [MTC]) or the thorax (specifically pul-
monary NET) (Figs. 4 and 5).1,8,56,57 Although positivity for
calcitonin is often used to support a diagnosis of MTC, pul-
monary NETs can also produce calcitonin (more precisely
(including bombesin and serotonin).1,8,58 Therefore, concur-
rent staining for monoclonal CEA is recommended. While
rare pulmonary NETs have been reported with variable CEA
positivity, diffuse CEA positivity is consistent with the diagno-
sis of MTC in a TTF-1-positive and calcitonin-positive
WDNET.1 Alternatively, positivity for bombesin supports a

Cancer Cytopathology December 2016 875

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Figure 4. Medullary thyroid carcinoma. The fine-needle aspiration biopsy from a lateral neck mass identified neuroendocrine tumor
cells with eccentric nuclei and a plasmacytoid appearance (A: Giemsa stain and B: Papanicolaou stain). The cell block was subjected
to a panel of markers. The tumor cells were diffusely positive for (C) chromogranin A, (D) thyroid transcription factor 1 (TTF-1), (E)
calcitonin, and (F) carcinoembryonic antigen. The cytomorphology and immunohistochemical findings were diagnostic of medullary
thyroid carcinoma.

diagnosis of pulmonary NET in the appropriate setting,
although most laboratories do not have access to this test.1,58
In cases where there is a need to distinguish between a pulmo-
nary versus thymic NET, positivity for TTF-1 favors a pulmo-
nary origin.59
At this point, it is important to emphasize that transcrip-
tion factors often lack lineage specificity in poorly differenti-
ated NECs, which can be a source of diagnostic error.1,8 In
particular, TTF-1 is commonly expressed in poorly differenti-
ated NECs regardless of the site of origin.1,8,60 However, in
some cases, the pathologist may be asked to exclude a Merkel
cell carcinoma from a metastatic NEC in the skin, given the
different treatment implications.1,7,8,61,62 In such cases, para-
nuclear dot-like positivity for CK20, Merkel cell polyoma
virus, and negativity for TTF-1 suggest the possibility of a
Merkel cell carcinoma.1,7,8,61,62

876 Cancer Cytopathology December 2016

 Algorithmic Approach to NETs in Targeted Biopsies/Duan and Mete

Figure 5. Pulmonary neuroendocrine tumor (NET). The fine-needle aspiration biopsy from the lung nodule identified a spindle cell
NET (A: Giemsa stain and B: cell block; H & E). The tumor cells were positive for (C) chromogranin A, (D) thyroid transcription factor 1
(TTF-1), and (E) CD56, and were negative for (F) carcinoembryonic antigen. The overall features were those of a pulmonary NET.

Various other NETs can arise in the head and neck
region, including pituitary and parathyroid neoplasms.1,63,64
As mentioned previously, paragangliomas can be found in this
location (ie, carotid body paragangliomas, thyroid paragan-
gliomas, laryngeal paragangliomas) and should be excluded
using epithelial markers and/or tyrosine hydroxylase. While
pituitary adenomas tend to arise in the sella turcica region,
they can invade into adjacent structures or present at ectopic
locations, including the nasopharynx, sphenoid wing, sphe-
noid sinus, nasal cavity, and temporal bone.1,65–67 Therefore,
if a pituitary adenoma is suspected, positivity for adenohypo-
physeal cell lineage transcription factors (pituitary-specific
transcription factor 1 [Pit-1], T-pit, and steroidogenic factor 1
[SF-1]) is helpful in confirming the adenohypophyseal cell ori-
gin.68,69 Parathyroid neoplasms can sometimes present as a
mass in the neck or mediastinum, or even as an intrathyroidal
mass.40,41,70,71 Parathyroid tumors express neuroendocrine
and epithelial markers as well as PTH, although the latter can
occasionally be expressed in some other NETs.40,41,72–75 Con-
sequently, positivity for GCM-2 and GATA-3 is helpful in

Cancer Cytopathology December 2016 877

Review Article
Figure 6. Metastatic enterochromaffin cell neuroendocrine tumor (NET) of midgut origin. The fine-needle aspiration and core needle
biopsy specimens from the liver lesions identified a well-differentiated NET. Diffuse positivity for (A) caudal type homeobox 2 (CDX-
2) and (B) serotonin suggested an enterochromaffin cell NET of midgut origin. Subsequent clinical and imaging workup confirmed the
presence of a primary NET of the small bowel.
the confirmation of parathyroid origin in a PTH-positive
NET.1,39–41,76
Intra-abdominal locations
The intra-abdominal cavity is a common source of NETs,
which can arise from the pancreas and various gastrointestinal
sites.1,7,17,77–79 In particular, primary pancreatic NETs are
important to recognize because they can be treated with newer
therapy regimens (including sunitinib and everolimus) and are
more sensitive to chemotherapy than NETs arising from other
sites.7,80–82 Furthermore, midgut NETs may be amenable to
octreotide therapy, with promising outcomes reported in a
subset of patients with advanced disease.7,83,84 In addition,
identification of primary small-bowel NETs (even in the pres-
ence of metastatic disease) may result in surgical intervention
to prevent further complications, including bowel obstruction,
ischemia, and perforation.1,7,79,85,86 Although the liver is a
common site of metastasis for NETs, it is important to note
that primary paraganglioma of the liver does exist.87,88 There-
fore, the identification of a NET in the liver is not always a
sign of metastatic disease. Furthermore, the presence of a
TTF-1-positive WDNET in the liver warrants further immu-
nohistochemistry, with calcitonin and monoclonal CEA, to
distinguish between a metastatic pulmonary NET versus a
metastatic MTC.
An immunohistochemical panel showing TTF-1 nega-
tivity, strong and diffuse staining for CDX-2, insulin gene
enhancer protein (ISL-1) negativity, and PDX-1-negativity in
a WDNET favors a midgut origin (usually jejunoileal or
appendiceal).1,8,17,89–96 CDX-2 is a nuclear transcription fac-
tor found in gastrointestinal epithelial cells distal to the stom-
ach.1,8,17,89–96 In particular, diffuse positivity for both
878
serotonin and CDX-2 is a characteristic feature of an entero-
chromaffin cell NET of midgut origin (Fig. 6).97 Although
WDNETs from the pancreas, foregut, and hindgut can also
express CDX-2 positivity, the staining pattern is usually weak
and patchy compared with the strong and diffuse staining
observed in midgut WDNETs.1,8,17,89–96 Therefore, assess-
ment for both extent and intensity of CDX-2 staining, along
with other hormones, increases the likelihood of a primary
midgut origin.1,8,17,89–96 Furthermore, it should be noted that
recent evidence suggests that the midgut (in particular the
ileum) is the most common primary site for metastatic
WDNETs of unknown primary.1,7,9,79
In contrast, an immunohistochemical panel demonstrat-
ing TTF-1 negativity, negative or patchy or weak staining for
CDX-2, ISL-1 positivity, and PDX-1 positivity suggests a
WDNET originating from the pancreas or duodenum.1,7,8
ISL-1 or Islet-1 is a transcription factor implicated in the dif-
ferentiation of neuroendocrine pancreatic cells, although it can
be found in chromaffin cells, calcitonin-producing parafollicu-
lar C cells, as well as the anterior/intermediate pituitary lobes
and some neurons involved in autonomic and endocrine con-
trol.1,8,98–102 In addition to pancreatic NETs, ISL-1 may be
expressed in NETs of duodenal and colorectal origin.1,8,102,103
When distinguishing between a pancreatic/duodenal
WDNET versus a colorectal WDNET, concurrent positivity
for PDX-1 is helpful to support a pancreatic/duodenal origin.
As its name implies, PDX-1 is a transcription factor impli-
cated in the development of the pancreas and duode-
num.1,8,104–107 Alternatively, demonstration of positivity for
prostatic acid phosphatase suggests a hindgut origin (usually
rectal NET; 82% positivity), with infrequent reports at non-
rectal sites (small bowel NETs; 20% positivity).1,8,77,108,109
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 Algorithmic Approach to NETs in Targeted Biopsies/Duan and Mete
Positivity for ISL-1 and CDX-2 (usually patchy/weak positiv-
ity) is also described in NETs of the hindgut.1,8,77,102 In a
ISL-1-positive and PDX-1-positive WDNET, the distinction
between a pancreatic versus a duodenal WDNET can be chal-
lenging. Immunohistochemical positivity for xenin, a hor-
mone peptide found in endocrine cells of the duodenum, may
be helpful to support a duodenal origin; however, this marker
is not available in the majority of laboratories.1,78,110 Although
PAX-8 is a nuclear transcription factor implicated in the devel-
opment of the optic, thyroid, urinary, and reproductive sys-
tems, it has also been shown to have strong
immunohistochemical expression in normal human islets and
pancreatic NETs.1,8,111–119 However, it should be noted that
these findings were primarily based on a commercially avail-
able polyclonal PAX-8 antibody that also identifies other
members of the PAX family (including PAX-3, PAX-5, and
PAX-6).1,8,111–119 In other words, it is likely that polyclonal
PAX-8 is cross-reacting with PAX-6 (which is highly expressed
in pancreatic islet cells), because PAX-8 messenger RNA
(mRNA) expression is generally low in islet cells.1,8,111–119,
Similar to xenin and prostatic acid phosphatase, other
hormonal markers may also help locate WDNETs within the
gastrointestinal tract.1,8,17 WDNETs of the foregut and
midgut often produce serotonin and substance P, whereas
WDNETs of hindgut origin tend to produce the peptide
spectrum of L cells, including glucagon-like peptide 1, pancre-
atic polypeptide, and peptide YY.1,17,77,97
Role of Predictive and Prognostic Biomarkers
In addition to the primary site and proliferation index of a
NET, several biomarkers with potential predictive and/or
prognostic usefulness have been described.1,8,75,120–127 The
majority of these assessments are performed on surgical speci-
mens. However, select markers may be considered on biopsy
specimens upon clinical request in specific presentations.
Although controversial, the detection and subtyping of
somatostatin receptors (SSTRs 1-5) by immunohistochemistry
is advocated to select patients for somatostatin analog therapy
and targeted peptide receptor radiotherapy.122,124–131 In addi-
tion, loss of expression for SSTR-2 (one of the common targets
of octreotide) correlates with poorer survival in patients with
gastroenteropancreatic NETs.17,124–128,132 In patients with
symptomatic NETs, tumor positivity for hormonal markers
may be of clinical use because those who develop secondary
endocrine syndromes tend to have increased morbidity and
mortality; furthermore, this approach may help identify
Cancer Cytopathology December 2016
potential circulating hormones to monitor for disease progres-
sion biochemically.1,75,121,123,133
In patients with pulmonary and gastrointestinal NETs,
loss of E-cadherin and b-catenin expression appears to be asso-
ciated with higher tumor grade.134,135 In those with pancreatic
NETs, immunohistochemical positivity for CEA-related cell
adhesion molecule 1 (CEACAM1), cytokeratin 19 (CK19),
and CD117 (C-kit) may be related to more aggressive behav-
ior and worse clinical outcomes.93,120,136–138 A recent study
also revealed that pancreatic NETs commonly harbor muta-
tions in multiple endocrine neoplasia type 1 [MEN1] (44%),
DAXX/ATRX (death domain-associated proteins; 43%), and
mechanistic target of rapamycin (mTOR) pathway genes
(14%).139 Subsequent studies reported that loss of DAXX,
ATRX, and phosphatase and tensin homolog (PTEN) expres-
sion are associated with worse survival in patients with pancre-
atic NETs.17,120,140,141 Negative progesterone receptor
expression may be associated with a worse prognosis.17,141
Expression of cyclooxygenase-2, p21, p18, and Rb have been
correlated with poorer survival in patients with gastroentero-
pancreatic NETs.17,132,142 In patients with hindgut NETs, the
distinction of L-cell phenotype from enterochromaffin cell ori-
gin using hormonal markers (glucagon-like peptide and pep-
tide YY) also appears to have prognostic implications.143,144
Various immunohistochemical markers associated with
genetic susceptibility have been reported in neuroendocrine
neoplasms. For instance, loss of p27 may suggest an underly-
ing germline CDKN1B mutation associated with multiple
endocrine neoplasia type 4 (MEN4) syndrome.40,145–149
However, one should also be aware of the relative frequency of
CDKN1B/p27 aberrancies in small bowel NETs, which can
lead to multifocal disease independent of MEN4 syn-
drome.150 Although loss of menin (the gene product of
MEN1) in pancreatic NETs may provide prognostic informa-
tion, it can help to select some patients for genetic testing of
MEN1 syndrome.139,145,148,151,152 However, it should be
noted that a significant number of NETs harbor somatic and
epigenetic alterations leading to reduced menin expression.139
In addition, extracolonic manifestations of Lynch syndrome
have also been expanded with the inclusion of various
NETs.153–155 Therefore, testing for mismatch repair proteins
is informative in the appropriate clinical context.
In patients with paragangliomas/pheochromocytomas,
loss of succinate dehydrogenase B (SDHB) expression is con-
sidered a harbinger of familial disease associated with any
SDH mutations.16,27,29–34,156,157 Furthermore, loss of SDHB
also provides prognostic data given the high frequency of
879
Review Article

Figure 7. Proposed algorithmic approach to neuroendocrine tumors (NETs) in targeted biopsies. -, negative immunohistochemical
expression; 1 , positive immunohistochemical expression; CDX-2, caudal type homeobox 2; CEA, carcinoembryonic antigen; ISL-1, insu-
lin gene enhancer protein (Islet-1); MTC, medullary thyroid carcinoma; PDNEC, poorly differentiated neuroendocrine carcinoma; PDX-1,
pancreatic and duodenal homeobox 1; PAP, prostatic acid phosphatase; SDHB, succinate dehydrogenase B; TTF-1, thyroid transcription
factor 1;WDNET, well-differentiated neuroendocrine tumor. *Evaluation of tumor proliferative activity depends on the anatomic site.

malignant disease associated with SDHB-driven paraganglio-
mas.16,27,29–34,156–158 Recent advances have also expanded the
spectrum of SDH-deficient neoplasms with the inclusion of
NETs.159,160 Similar to paragangliomas and pheochromocyto-
mas, the use of SDHB immunohistochemistry may be consid-
ered to predict SDH-related pathogenesis in other
neuroendocrine neoplasms.160
Conclusions
Over the past decade, our understanding of the clinical spec-
trum and pathogenesis of NETs has grown substantially with
advances in molecular and cellular pathology. As a result, the
management of these tumors has become increasingly com-
plex, with greater emphasis on the diagnostician’s role in guid-
ing therapy.161 For instance, the treatment of patients with
advanced WDNETs is increasingly dependent on the site of
origin of the tumor.7,10 However, there are still clinical presen-
tations in which the primary site remains unknown prior to
the pathological examination (ie, in a biopsy of a liver or
lymph node metastasis).7,8 In conjunction with clinical and
morphological findings, immunohistochemistry has become
an indispensable ancillary tool to help solve this clinical conun-
drum. As a general rule, a stepwise algorithmic approach (with
applications of immunohistochemical markers) is advised to
avoid diagnostic errors (Fig. 7). In patients with a suspected
NET, we recommend to: 1) start by confirming neuroendo-
crine differentiation, using chromogranin A and synaptophy-
sin concurrently; 2) consider the diagnostic category of
paraganglioma in a keratin-negative and transcription factor-
negative neuroendocrine neoplasm (positivity for tyrosine
hydroxylase confirms a diagnosis of paraganglioma); and 3)
distinguish a WDNET from a poorly differentiated NEC by
assessing cytomorphology and/or degree of proliferation, using
the Ki-67 labeling index. In a WDNET of unknown primary,
we suggest an initial panel that combines transcriptional fac-
tors (TTF-1, CDX-2, ISL-1, and PDX-1) along with hor-
mone and structural biomarkers as deemed necessary to
determine the site of origin; depending on the primary site
and clinical indications, additional biomarkers may be per-
formed to provide prognostic information to guide therapy
and/or screen patients for genetic testing.
FUNDING SUPPORT
No specific funding was disclosed.

880 Cancer Cytopathology December 2016

 Algorithmic Approach to NETs in Targeted Biopsies/Duan and Mete
CONFLICT OF INTEREST DISCLOSURES
The authors made no disclosures.
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Cancer Cytopathology December 2016