REVIEW ARTICLE
Assigning Site of Origin in Metastatic Neuroendocrine
Neoplasms: A Clinically Significant Application
of Diagnostic Immunohistochemistry
Andrew M. Bellizzi, MD*w
Abstract: The neuroendocrine epithelial neoplasms (NENs) include
well-differentiated neuroendocrine tumors (WDNETs) and poorly
differentiated neuroendocrine carcinomas (PDNECs). Whereas
PDNECs are highly lethal, with localized Merkel cell carcinoma
somewhat of an exception, WDNETs exhibit a range of “indolent”
biologic potentials—from benign to widely metastatic and even-
tually fatal. Within each of these 2 groups there is substantial
morphologic overlap. In the metastatic setting, the site of origin of
a WDNET has significant prognostic and therapeutic implications.
In the skin, Merkel cell carcinoma must be distinguished from
spread of a visceral PDNEC. This review intends to prove the thesis
that determining the site of origin of a NEN is clinically vital and
that diagnostic immunohistochemistry is well suited to the task. It
will begin by reviewing current World Health Organization ter-
minology for the NENs, as well as an embryologic and histologic
pattern–based classification. It will present population-based data
on the relative frequency and biology of WDNETs arising at var-
ious anatomic sites, including the frequency of metastases of
unknown primary, and comment on limitations of contemporary
imaging techniques, as a means of defining the scope of the prob-
lem. It will go on to discuss the therapeutic significance of site of
origin. The heart of this review is a synthesis of data compiled from
>100 manuscripts on the expression of individual markers in
WDNETs and PDNECs, as regards site of origin. These include
proteins that are considered “key markers” and others that are
either useful “secondary markers,” potentially very useful markers
that need to be further vetted, or ones that are widely applied
despite a lack of efficacy. It will conclude with my approach to the
metastatic NEN of unknown origin.
Key Words: well-differentiated neuroendocrine tumor, poorly dif-
ferentiated neuroendocrine carcinoma, carcinoid tumor, Merkel
cell carcinoma, carcinoma of unknown primary, immunohis-
tochemistry, transcription factor, CDX2, TTF-1, cytokeratin 20
(Adv Anat Pathol 2013;20:285–314)
T he neuroendocrine epithelial neoplasms (NENs),
including well-differentiated neuroendocrine tumors
(WDNETs) and poorly differentiated neuroendocrine car-
cinomas (PDNECs), are characterized by the expression of
keratin intermediate filaments and the production of pep-
tide hormones and/or biogenic amines. Whereas PDNECs
are biologically aggressive and highly lethal, with localized
Merkel cell carcinoma (MCC) somewhat of an exception,
From the *Department of Pathology, University of Iowa Hospitals and
Clinics; and wDepartment of Pathology, University of Iowa Carver
College of Medicine, Iowa City, IA.
The author has no funding or conflicts of interest to disclose.
Reprints: Andrew M. Bellizzi, MD, Department of Pathology, Uni-
versity of Iowa Hospitals and Clinics, 200 Hawkins Drive, Iowa
City, IA 52242 (e-mail: andrew-bellizzi@uiowa.edu).
Copyright r 2013 by Lippincott Williams & Wilkins
Adv Anat Pathol  Volume 20, Number 5, September 2013
WDNETs exhibit a range of “indolent” biologic
potentials—from benign to widely metastatic and even-
tually fatal. Within each of these 2 groups of tumors, there
is substantial morphologic overlap. The PDNECs are
composed of “small,” intermediate, or large cells, often
exhibit diffuse growth patterns, and are highly proliferative.
The WDNETs, regardless of site of origin, are charac-
terized by a series of growth patterns collectively referred to
as “organoid” and frequently demonstrate finely granular
chromatin. The range of biologies referenced above for the
WDNETs can to a large extent be predicted based on
anatomic site. In addition, the therapeutic armamentarium
for metastatic WDNETs is rapidly expanding, and the
efficacy of individual agents is related to site of origin.
When faced with a metastatic WDNET of unknown
origin (generally to the liver), an all-too-familiar diagnostic
scenario, pathologists have long been content to levy a
diagnosis to the effect of “metastatic neuroendocrine
tumor” and to move on with their day. This practice is no
longer sufficient. Patients and their treating surgical oncolo-
gists, endocrinologists, and medical oncologists deserve
some attempt at assigning site of origin, as well as comment
on the degree of proliferation. These parameters are of signi-
ficant prognostic and therapeutic importance. In the skin,
MCC must be distinguished from spread of a visceral
PDNEC. Most pathologists are familiar with the application
of cytokeratin (CK) 20 and thyroid transcription factor-1
(TTF-1) immunohistochemistry (IHC) in this setting. Fewer
are familiar with the frequency of TTF-1 expression in
PDNECs, regardless of site of origin. At least 2 other
markers are useful in this setting, including one that directly
reflects the etiopathogenesis of MCC.
Regarding the WDNETs, a number of markers,
including several transcription factors, have emerged that
are very useful in assigning site of origin. Most of these
antibodies are “workhorses” in diagnostic IHC (eg, TTF-1,
CDX2), others are emerging “powerhouses” (eg, polyclonal
PAX8), and a few others are “classics” [eg, progesterone
receptor (PR), S-100], perhaps with a “new trick up their
sleeves.” Pathologists are, in general, unfamiliar with this
application of IHC or will make assumptions about the
expression pattern of a marker based on data from ade-
nocarcinoma (eg, they assume that CDX2 will be expressed
by tumors throughout the intestine).
This review intends to prove the thesis that determin-
ing the site of origin of a NEN is clinically vital and that
diagnostic IHC is well suited to the task. It will begin by
reviewing current World Health Organization (WHO) ter-
minology as well as an embryology-based and a histologic
pattern–based classification that I find useful in organizing
my thoughts about these lesions. It will present (1) pop-
ulation-based data on the relative frequency and biology of
WDNETs arising at various anatomic sites, as a framework
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Bellizzi
for, in the metastatic setting, conceptualizing the pretest
probabilities of spread from various sites; (2) the frequency
of metastases of unknown primary in several recent series;
and (3) the limitations of current imaging techniques, as a
means of defining the scope of the problem. It will go on to
discuss the contemporary therapeutic significance of site of
origin. The heart of this review is a synthesis of data com-
piled from >100 manuscripts on the expression of indivi-
dual markers in WDNETs and PDNECs, as regards site of
origin. These will include proteins I consider “key markers”
and others that are either useful “secondary markers,”
potentially very useful markers that need to be further vetted,
or ones that are widely applied despite a lack of efficacy in
this setting. It will conclude with my approach to the meta-
static NEN of unknown origin.
TUMOR CLASSIFICATIONS
World Health Organization Classification
The WHO Classification of Tumours of the Digestive
System presents a nomenclature for gastroenteropancreatic
(GEP) NENs that supplants the WHO 2000 Classification.1
Morphologically well-differentiated NENs are referred
to simply as “neuroendocrine tumors” (NETs), whereas
poorly differentiated examples are classified as “neuro-
endocrine carcinomas” (NECs). The “well-differentiated
endocrine carcinoma” of WHO 2000 no longer exists, with
the current classification of a NEN based on its appearance,
rather than whether or not it has metastasized. In this
classification, the term “NET” implies a well-differentiated
tumor (although WDNET will be used often throughout
this review for clarity).
As discussed above, NETs are biologically hetero-
genous, and it is well recognized that a group of tumors
characterized by tumor necrosis, increased mitotic activity,
and increased tumor fraction participating in the cell cycle
[ie, increased proliferation index (PI); determined by the
percentage of tumor cells expressing Ki-67 by IHC] carve
out a “middle path” between less-proliferative NETs and
PDNECs. In WHO 2010, this is captured in the grade of a
NET. By definition, grade (G)1 tumors demonstrate <2
mitotic figures per 10 high-power fields (HPF) and/or a Ki-
67 index of r2%, whereas G2 is assigned on the basis of a
mitotic count of 2 to 20/10 HPF and/or a Ki-67 index of
3% to 20%. The NECs can be described as being of “large
cell” or “small cell type” (the WHO 2010 term “NEC”
implies a poorly differentiated tumor, although PDNEC
will be used for clarity). NECs are G3, with a mitotic count
>20/10 HPF and/or a Ki-67 index of >20%.
The most recent WHO Classification of pulmonary
NENs is contained in the 2004 WHO Classification of
Tumours: Pathology and Genetics of Tumours of the Lung,
Pleura, Thymus and Heart.2 In this system, the term “carcinoid
tumor” persists. WDNETs of the lung are classified as “typical
carcinoid” (TC) or “atypical carcinoid” (AC) on the basis of
mitotic rate and the presence or absence of tumor necrosis.
TCs possess well-differentiated histology, demonstrate <2
mitotic figures per 2 mm2 (generally related as 10 HPF but
actually closer to 8 HPF on many modern microscopes), lack
necrosis, and measure Z0.5 cm. A diagnosis of AC is rendered
when a morphologically similar tumor contains 2 to 10 mitotic
figures per 2 mm2 and/or necrosis.
It is noteworthy that Ki-67 PI is not presently a
component of the classification of pulmonary NENs
(although it may be part of the next one). Despite this fact,
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Adv Anat Pathol  Volume 20, Number 5, September 2013
Ki-67 IHC has been shown to highlight PIs for TC and AC
analogous to those seen with G1 and G2 GEP NETs,
respectively (reviewed in Rekhtman3). As has been pointed
out by several authors, Ki-67 IHC is often a useful diag-
nostic adjunct in these tumors, particularly in crushed small
biopsies in which mistaking a carcinoid tumor for small cell
lung carcinoma (SCLC) is a genuine pitfall of serious
clinical significance.3–5
The PDNECs include SCLC and large cell neuro-
endocrine carcinoma (LCNEC). To qualify as poorly dif-
ferentiated, a pulmonary NEN must demonstrate at least
11 mitotic figures per 2 mm2, although examples usually
exhibit much higher mitotic rates. [It is noteworthy that the
current mitotic “floor” for a pulmonary PDNEC (Z11/
2 mm2) is lower than that for a GEP NEC (> 20/10 HPF).]
Compared with SCLC, LCNEC is more apt to demonstrate
an organoid (rather than diffuse) architecture, is composed
of larger cells with more abundant cytoplasm, typically
contains prominent nucleoli, and may or may not exhibit
“neuroendocrine chromatin.” Because of substantial mor-
phologic overlap with large cell (undifferentiated) carcinoma, a
diagnosis of LCNEC formally requires the immuno-
histochemical demonstration of expression of at least 1 gen-
eral neuroendocrine marker.2
Although WDNETs and PDNECs predominate in the
GEP system and lung, respectively, similar-appearing
tumors occur in every organ system. These have generally
been classified by appropriating the terminology used for
GEP or pulmonary tumors. As most of the relevant WHO
Blue Books are still in their third editions, the descriptor
“carcinoid tumor” (a term emphatically stricken from the
record for GEP NETs) still abounds. At some sites
WDNETs are all classified as well-differentiated NECs,
based on the premise that all WDNETs are potentially
malignant.6 These semantics should not interfere with the
simple facts that the NENs, regardless of anatomic site, can
be separated into well-differentiated, generally indolent
tumors and poorly differentiated, aggressive ones, and that,
for the well-differentiated tumors, evidence of increased
cell turnover suggests the possibility of more aggressive
behavior. A couple tumors deserve special mention here.
MCC is a PDNEC of the skin, and medullary thyroid
carcinoma is defined as a “malignant tumor of the thyroid
gland showing C-cell differentiation.”7
Williams and Sandler Classification
Based on observations of the histology, histochemical
features, biochemical content, and biology of WDNETs,
Williams and Sandler,8 in an article published in The Lancet
in 1963, proposed that tumors be grouped into those that
are foregut, midgut, and hindgut derived. The familiar
“midgut carcinoid” is characterized by a nested archi-
tecture, often contains brightly eosinophilic cytoplasmic
granules that are argentaffin positive (ie, they reduce silver
salts to metallic silver), has a high 5-hydroxytrypamine
(serotonin) content, and is frequently associated with car-
cinoid syndrome. Foregut-derived and hindgut-derived
tumors share several characteristics that distinguish them
from midgut ones. They have a tendency toward trabecular
architecture, are nonargentaffin, and contain little, if any,
serotonin. Compared to hindgut tumors, foregut ones are
more apt to be associated with carcinoid syndrome and
often secrete 5-hydroxytryptophan.
Transcription factor expression is tightly regulated in
time and space and drives embryonic patterning. Thus, the
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Adv Anat Pathol  Volume 20, Number 5, September 2013 Neuroendocrine Neoplasms: Assigning Site of Origin

FIGURE 1. Architectural patterns in well-differentiated neuroendocrine tumors: Soga and Tazawa classification. A, Type A tumors are
nested; this histology is typical of ileal tumors. B, Type B tumors grow in ribbon-like fashion, as in this rectal tumor. C, Type C tumors are
composed of tubular, acinar, or rosette-like structures. This example was located in the ampulla, and it had been initially diagnosed on
biopsy as an adenocarcinoma. Note, also, the 2 psammomatous calcifications in the upper portion of this image. D, Type D tumors have
“diffuse” architecture. This appendiceal tumor, which was metastatic at presentation, had type A architecture elsewhere, and, in fact,
mixed patterns are often seen (hematoxylin and eosin, each 200).

Williams and Sandler classification provides an intuitively
appealing framework upon which to hang the observation
that jejunal, ileal, and appendiceal WDNETs, all midgut
derived, cluster together in terms of transcription factor
expression and that pancreaticoduodenal tumors, foregut
derived and arising from intimately developmentally related
organs, would, by transcription factor expression patterns, be
essentially indistinguishable. Similarly, the classification may
be useful to predict the expression profiles of cecal and other
colonic tumors, for which little data exist. However, it is
insufficient to explain all observations—for example, why the
transcription factor signature of pancreaticoduodenal tumors
seems to have more in common with hindgut-derived rectal
tumors than with fellow foregut-derived gastric ones.
Soga and Tazawa Classification
Soga and Tazawa,9 in a manuscript published in Cancer
in 1971 (based on their observations in 62 tumors), described
5 architectural patterns in “carcinoid tumors” (types A to D
and mixed) and correlated them with site of origin (Figs. 1A–
D). Type A tumors are composed of solid nests (ie, insular).
Type B tumors are trabecular or “ribbon-like,” with the
cords of cells having a tendency to anastomose. Type C
tumors contain tubular, acinar, or rosette-like structures (ie,
pseudoglandular). Type D tumors exhibit “lower or atypical
differentiation” (ie, “diffuse” or “undifferentiated”). Mixed
tumors exhibit any combination of patterns A to D. In their
series, type A histology was typical of “midgut carcinoids,”
foregut-derived tumors tended to be type B, and hindgut
tumors were apt to be mixed.
In the Armed Forces Institute of Pathology (AFIP)
series of 167 jejunoileal WDNETs, 93% were type A.10 The
association with type A histology is less strong in the
appendix, where Burke et al11 described, among 86 pure
WDNETs, 50% type A and 24% mixed tumors, as well as
20% “tubular” carcinoids and 6% “clear cell” carcinoids.
While the type A, mixed, and clear cell tumors were
argentaffin positive and produced serotonin [characteristics

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Bellizzi Adv Anat Pathol  Volume 20, Number 5, September 2013

of enterochromaffin (EC)-cell WDNETs], the tubular car-

cinoids reacted with antibodies to glucagon. The latter TABLE 1. Site of Origin and Outcome in 35,825 Well-
Differentiated Neuroendocrine Tumors From the Surveillance,
tumor type may be related to rare appendiceal WDNETs Epidemiology, and End Results Registry (1973 to 2004)
composed of L cells (enteroglucagon cells), which express
glucagon-like peptides and peptide YY; these minute, Relative Distant Median
indolent tumors are distinctly trabecular (type B).12 Small, Frequency Metastasis Survival
incidentally discovered rectal WDNETs are also charac- Site of Origin (%)* (%) (mo)
teristically composed of L cells and demonstrate a type B Foregut
growth pattern.13 Type C histology is typical of somatos- Lung 27 28 193
tatin-producing D-cell tumors of the duodenum.14 Gar- Thymus 0.4 31 77
brecht et al15 found that, among 82 non-multiple endocrine Stomach 6.0 15 124
neoplasia type 1 (MEN1)-related duodenal NENs, 26% Duodenum 3.8 9 99
predominantly or exclusively expressed somatostatin; Pancreas 6.4 64 42
Liver 0.8 28 23
among these, 60% demonstrated at least a component of Midgut
type C growth. Also of note, 71% of these D-cell tumors Jejunum/ileum 13 30 88
occurred in the ampulla and 58% contained psammoma Appendix 3.0 12 NR
bodies. A type C component was noted in only 22% of Cecum 3.2 44 83
pancreatic D-cell tumors, which were much less common Colon 4.0 32 121
than in the duodenum (4%; 21/541). Outside of the jeju- Hindgut
noileum (type A) and rectum (type B), WDNETs are often Rectum 17 5 240
mixed.9,16 Other/unknown 15 NA NS
primary
Data from Yao et al.17
FREQUENCY, BIOLOGY, AND IMAGING *Data in this column are from the period between 2000 and 2004.
OF WELL-DIFFERENTIATED NEUROENDOCRINE NA indicates not applicable; NR, not reached; NS, not stated.
TUMORS
Population-based Data Regarding Relative
Frequency and Biology
In 2008, Yao et al17 published an analysis of the Notable among these studies is one from Kirshbom
epidemiology of WDNETs based on 35,825 tumors reported et al20 that reported on the clinical and biochemical char-
to the Surveillance, Epidemiology, and End Results (SEER) acteristics of 143 metastatic WDNETs of unknown primary
Program from 1973 to 2004. This study expanded on pre- (among >750 total WDNETs) seen by J.M. Feldman at
vious reports of US population–based cohorts published in Duke University Medical Center/Durham Veterans Affairs
1975 and 2003.18,19 Data from this study regarding relative Hospital over a nearly 30-year period. Patients with
tumor frequency, proportion presenting with distant meta- unknown primary tumors had high levels of urine 5-
stasis, and median overall survival—segregated by anatomic hydroxyindoleacetic acid (main serotonin metabolite) and
site—are summarized in Table 1. The 5 most frequent pri- urine, platelet, and serum serotonin levels on par with
mary sites included lung (27%), rectum (15%), jejunoileum those seen in patients with metastatic midgut WDNETs
(13%), pancreas (6.4%), and stomach (6%), accounting for
two thirds of all tumors. Among these, pancreatic tumors
most often presented with distant metastasis (64%) and were
associated with the shortest median survival (42 mo). By TABLE 2. Proportion of All WDNETs Accounted for by Metastases
comparison, jejunoileal tumors were metastatic half as often From Unknown Primaries in Recent Series
(30%) and associated with twice as long overall survival Frequency
(88 mo). Lung tumors were metastatic at a rate of 28% but of Total
were associated with long survival (193 mo), only exceeded Unknown Number
by rectal (240 mo) and appendiceal (median survival not Primaries of
reached) tumors. A significant weakness of the SEER data References (%) WDNETs Comments
set is that it relies on the reporting of tumors to the registry, Kirshbom 19 > 750 US single-center cohort
with the authors acknowledging that “many small, benign- et al20
appearing tumors likely are excluded.” This may explain why Yao et al17 13 35,825 US population–based
appendiceal tumors, for example, perceived as relatively cohort
common among WDNETs, represented only 3%. Pape et al21 10.5 399 German single-center
cohort; only
Frequency and Biology of Metastatic Tumors of gastroenteropancreatic
Lombard- 11.5 668 French multicenter
Unknown Origin Bohas cohort; only
Data from recent studies reporting the proportion of et al22 gastroenteropancreatic
metastatic tumors of unknown origin among all WDNETs Garcia- 9 907 Spanish population–based
are summarized in Table 2. The frequency has ranged from Carbonero cohort; only
9% (population-based cohort)23 to 19% (single-center, et al23 gastroenteropancreatic
referral-based cohort).20 The median of these studies is Catena et al24 11 750 Italian single-center
11%. In the SEER series, the frequency of tumors of cohort
“other/unknown” origin was exceeded only by that of WDNET indicates well-differentiated neuroendocrine tumor.
bronchopulmonary and rectal primaries.17

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Adv Anat Pathol  Volume 20, Number 5, September 2013
(and greater than those seen in patients with locoregional
midgut tumors and much greater than patients with foregut
or hindgut tumors). The survival curves for unknown pri-
mary and metastatic midgut tumors were also quite similar,
with 10-year survivals of 22% and 28%, respectively, and
convergence of survival around 15 to 20 years. These
findings led the authors to suggest that most WDNETs of
unknown origin may represent metastases from midgut
primaries.
A retrospective analysis of the University of Cal-
ifornia, San Francisco (UCSF) experience with liver
metastases from WDNETs lends additional support to
Kirshbom and colleagues’ hypothesis.25 Wang and colleagues
identified 123 patients diagnosed between 1993 and 2008,
in whom hepatic metastasis was the initial disease pre-
sentation in 64% (n = 79). Among 71 patients presenting
with metastatic disease, adequate medical records, and in
whom a workup was undertaken, the primary tumor was
successfully localized [making use of various combinations
of computed tomography (CT), somatostatin receptor
imaging (SRI), positron emission tomography (PET),
magnetic resonance imaging, and upper and lower endos-
copy] in 56 (79%): pancreas 27, small intestine 17, colon 7,
lung 2, stomach 1, kidney 1, presacral 1. The remaining 15
patients underwent surgical exploration to detect an occult
primary, which successfully identified 12 ileal and 1 jejunal
tumor (13/15; 87%).
Imaging of Well-Differentiated Neuroendocrine
Tumors
In addition to conventional imaging modalities,
WDNETs can be visualized using nuclear medicine tech-
niques that take advantage of distinctive biochemical
properties of this class of tumors, including increased glu-
cose metabolism, uptake of biogenic amines, and frequent
expression of somatostatin receptors (SSTRs). The first
of these is the basis of PET imaging with the radiotracer
18
F-fluoro-2-deoxy-D-glucose, which is more pertinent to
the highly metabolically active PDNECs. The uptake of
radioiodine-labeled metaiodobenzylguanidine (MIBG) by
the majority of WDNETs is the basis of the MIBG scan, as
well as MIBG-radionuclide therapy. Although this modal-
ity has been overtaken by SRI in NENs, MIBG-radio-
nuclide therapy remains a mainstay of the treatment of
advanced pheochromocytoma/paraganglioma.26
Somatostatin is an inhibitory peptide hormone pro-
duced in regions of the brain and by D cells in the stomach,
intestine, and pancreas. In the gastrointestinal (GI) tract,
somatostatin suppresses the release of several hormones
including gastrin, cholecystokinin, vasoactive intestinal
peptide, insulin, and glucagon. It mediates its effects
through SSTRs, which exist as 6 subtypes (SSTR1, 2A, 2B,
3 to 5). Frequent SSTR expression by NENs is the basis of
the OctreoScan, in which the somatostatin analogue
octreotide is coupled to 111In through the chelator dieth-
ylene triamine pentaacetic acid. Of the SSTR subtypes,
a positive scan has been shown to most closely relate to
SSTR2A expression,27,28 which can be detected immuno-
histochemically in 60% to 90% of NENs (see below).29–34
Newer radiotracers couple octreotide or octreotate to
68
Ga via 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetra-
acetic acid (DOTA) (eg, DOTATOC, DOTANOC,
DOTATATE). These are used with PET and hybrid PET/
CT imaging (as opposed to planar imaging or single
photon–emission CT with the 111In compound) and hold
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Neuroendocrine Neoplasms: Assigning Site of Origin
promise for superior spatial resolution, decreased patient
exposure to radiation, and shorter scanning times.35,36
A common response to the suggestion that patholo-
gists should participate in assigning the site of origin of a
metastatic WDNET of unknown primary is that we should
“let radiology do it.” In the UCSF experience discussed
above, although CT successfully detected pancreatic
WDNETs (pancNETs) in every case, the sensitivities of CT
and SRI (presumably OctreoScan) for detecting tubal gut
primaries were only 34.6% and 26.2%, respectively.25
Savelli et al37 found that among 36 patients presenting with
a metastatic NET of unknown primary (among 428 GEP
NETs; 8.4%), OctreoScan successfully localized the pri-
mary in 39% (9 midgut, 3 pancreas, 1 cecum, 1 colon).
More recently, Prasad et al35 reported that DOTANOC
PET/CT identified a primary site in 59% of 59 patients (14
jejunoileum, 16 pancreas, 2 colorectum, 2 lung, 1 para-
ganglioma). Thus, traditional SRI may be useful in
assigning the primary site in less than half of cases. Newer
PET tracers appear more sensitive, but they are not widely
available. An additional consideration is cost. OctreoScan
is available at most US hospitals with a Nuclear Medicine
section, is charged at about $8000, and demonstrates a
sensitivity of 20 mm (the mean size of the occult small
intestinal primaries detected by surgical exploration in
Wang and colleagues’ series was 13.8 mm). In contrast,
DOTA scans, with a sensitivity of 4 mm, are, at present,
available at only 4 US centers, through the Food and Drug
Administration’s Investigational New Drug program (T.M.
O’Dorisio, written personal communication). Diagnostic
IHC is nearly universally available and is much less
expensive than either of these imaging techniques (or a
high-resolution CT for that matter); the Medicare Fee
Schedule for Current Procedural Terminology code 88342
is around $100.38
THERAPEUTIC SIGNIFICANCE OF DETERMINING
THE SITE OF ORIGIN IN WELL-DIFFERENTIATED
NEUROENDOCRINE TUMORS
Key therapeutic determinants in WDNETs include
tumor site and size, anatomic extent of disease (local,
regional, metastatic), presence of a functional syndrome,
and, in metastatic disease not amenable to surgery with
curative intent, site of origin of the primary tumor. Ther-
apeutic options include endoscopic removal (for select
gastroduodenal and rectal tumors), surgery, ablative/
locoregional techniques (for liver metastases), antisecretory
treatment (for functioning tumors), and antiproliferative
treatment. In patients with locoregional disease, the tumor
is generally excised. Metastatic disease to the liver,
depending on the pattern and extent of spread, is also
potentially resectable.39–42 Adjuvant therapy following an
R0 or R1 resection is not standard of care.43 Patients with
diffuse hepatic involvement or extrahepatic disease are
candidates for antiproliferative therapy. Even patients with
advanced metastatic disease may have their primary tumor
resected; this is particularly the case for jejunoileal tumors,
which have a tendency to cause obstruction, bleeding, and
ischemia (due to mesenteric vascular elastosis).
Antiproliferative treatment options are very different
in metastatic midgut and pancNETs (Table 3). Classes of
agents include those also used in antisecretory therapy,
systemic chemotherapeutics, small molecular inhibitors,
and peptide receptor–targeted radiotherapeutics. The
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Bellizzi
TABLE 3. Options for Antiproliferative Medical Therapy in
Metastatic Neuroendocrine Tumors
Applicable in
Agent Mechanism Midgut Pancreatic
Octreotide Somatostatin analogue + +
Interferon-a Immune activation + +
Streptozocin Alkylating agent +
Temozolomide Alkylating agent +
Everolimus mTOR inhibitor +/ + CDX2, one of 3 human homologues of the Drosophila
Sunitinib Tyrosine kinase inhibitor + gene Caudal, is a homeodomain-containing transcription
somatostatin analogue octreotide is the mainstay of anti-
secretory therapy in functioning tumors (including patients
with carcinoid syndrome); interferon-a is a second-line
agent due to its unfavorable side-effect profile. These agents
also have weak antiproliferative activity and are utilized in
advanced metastatic disease of midgut origin.44,45 Metastatic
pancNETs often respond to streptozocin-based systemic
chemotherapy.46 Alternatively, temozolomide, another alky-
lating agent, may be used.47 Intriguingly, in 1 retrospective
study, immunohistochemically detectable deficiency of the
DNA-repair enzyme O(6)-methylguanine DNA methyl-
transferase, observed in half of pancreatic and in no jeju-
noileal tumors, predicted treatment response.48 The response
rate of midgut tumors to both of these agents has been shown
to be poor.48,49
There is surging clinical interest in directed biological
therapy. Small-molecule inhibitors have been increasingly
evaluated in clinical trials. The mTOR inhibitor everolimus
was recently shown in a phase 3 randomized control trial to
prolong progression-free survival in patients with advanced
pancNETs by 6.4 months (vs. best supportive care).50
A similar trial in advanced midgut tumors fell just short of
reaching statistical significance, but given the paucity of
therapeutic alternatives, everolimus may be considered.51
A phase 3 trial of the multiple receptor tyrosine kinase
inhibitor sunitinib (inhibits platelet-derived growth factor
receptors, vascular endothelial growth factor receptors, and
KIT, among others) in advanced pancNETs was similarly
positive,52 while efficacy was not demonstrated in midgut
tumors.53 Finally, although peptide receptor–targeted ther-
apy (eg, 90Y-DOTATOC) holds promise in both advanced
midgut and pancreatic tumors, it is not widely available in the
United States.54,55
THERAPEUTIC SIGNIFICANCE OF DETERMINING
THE SITE OF ORIGIN IN POORLY
DIFFERENTIATED NEUROENDOCRINE
CARCINOMAS
In the skin, the critical distinction is between MCC and
metastatic visceral PDNEC. Clinically localized MCC is
treated with excision and sentinel lymph node biopsy.56 A
positive sentinel lymph node biopsy prompts completion
lymphadenectomy and/or radiation therapy. The primary
tumor bed may also be treated with radiation therapy,
especially in patients considered at high risk for local recur-
rence (eg, positive margins, large tumor size, lymphovascular
space invasion). At other potentially metastatic sites (eg,
liver), determination of the site of origin of a PDNEC
is mainly of academic interest, as all metastatic tumors,
regardless of origin, are treated with platinum-based
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Adv Anat Pathol  Volume 20, Number 5, September 2013
“SCLC” chemotherapy (ie, cisplatin or carboplatin and
etoposide).57–59
KEY MARKERS IN DETERMINING THE SITE OF
ORIGIN OF A WELL-DIFFERENTIATED
NEUROENDOCRINE TUMOR
Caudal type Homeobox 2 (CDX2)
factor critical in gut development and patterning and in the
maintenance of its phenotype.60,61 It is normally expressed
by epithelial cells of the tubal gut distal to the stomach.62
CDX2 expression can also be found in pancreatic cen-
troacinar and intercalated and intralobular duct cells and in
scattered cells in the larger ducts.63 Downstream targets
include MUC2, sucrose-isomaltase, and lactase. Induction
of CDX2 expression is characteristic of gastric intestinal
metaplasia64 and Barrett’s esophagus.65 Cdx2 homozygous–
null mouse embryos fail to implant, as Cdx2 expression is
required for the development of trophoectoderm. In a con-
ditional knockout (that overcomes the implantation block)
the tubal gut, which is lined by squamous instead of colum-
nar epithelium, ends in a blind pouch at the cecum.66
Diagnostic pathologists routinely use CDX2 as a
marker of intestinal-type adenocarcinomas. Aside from
colorectal and appendiceal tumors, in which expression is
detected in most cases (> 90%), it is often, although var-
iably, reported in tumors arising elsewhere in the gut and in
other tumors with intestinal-type differentiation (eg, muci-
nous ovarian tumors).63,67–69 Two early IHC surveys of
CDX2 expression reported positivity in a significant fraction
of intestinal WDNETs.63,68 Subsequently, CDX2 expression
has been shown to be highly sensitive and fairly specific for a
midgut origin (ie, jejunoileum or appendix). Nearly all of the
data regarding CDX2 expression in WDNETs are based on
staining with the mouse monoclonal antibody (MoAb)
CDX2-88. A recent study demonstrated high sensitivity and
superior specificity for a jejunoileal origin (appendiceal
tumors were not studied) with the rabbit MoAb EPR2764Y.70
Data from 14 studies assessing CDX2 expression
in primary and metastatic WDNETs are summarized
in Table 4. Expression has been detected in 90% (177/197)
of primary and 91% of metastatic (129/142) jejunoileal
tumors (Figs. 2A, B). Similarly, CDX2 positivity has been
noted in 93% (67/72) of appendiceal primaries; only a
handful of metastases have been tested (83%; 5/6). CDX2
expression has been detected in 31% (14/45) of duodenal,
14% (10/74) of gastric, and 29% (32/110) of rectal pri-
maries. A large number of pancreatic tumors have been
assessed, with CDX2 expression found in 16% (66/415) of
primary and 18% (15/84) of metastatic tumors. Only 3%
(8/233) of pulmonary carcinoids have been positive. The
specificity of CDX2 in this diagnostic context is probably
increased by taking quantity and intensity of staining into
account. Whereas midgut tumors generally demonstrate
diffuse, strong staining, expression in the foregut and
hindgut GEP tumors is often weak and patchy.
One additional anatomic site deserves special mention
here. WDNETs of the ovary often arise in the background
of a teratoma and are divided into insular, trabecular,
strumal, and mucinous types. Rabban et al83 found that 5
of 7 (71%) ovarian WDNETs with an insular growth pat-
tern expressed CDX2, while none of 8 (0%) tumors with a
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TABLE 4. CDX2 Expression in Well-Differentiated Neuroendocrine Tumors

n (%)
References Antibody Lung Stomach Duodenum Pancreas Jejunum/Ileum Appendix Cecum Colon Rectum
Moskaluk et al63 CDX2-88 P: 2/7 (29) P: 0/3 (0) P: NE P: 4/14 (29) P: ND P: ND P: NE P: NE P: 7/9 (78)
M: NE M: NE M: NE M: NE M: NE M: NE M: NE M: NE M: NE
Barbareschi et al71 CDX2-88 P: 0/30 (0) P: 5/5 (100) P: 4/4 (100) P (F): 0/13 (0) P: 14/14 (100) P: 6/6 (100) P: NE P: 0/1 (0) P: 7/9 (78)
M: 0/1 (0) M: 1/1 (100) M: 1/1 (100) P (NF): 14/48 (29) M: 6/6 (100) M: NE M: NE M: NE M: NE
M: 1/6 (17)

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Erickson et al72 CDX2-88 P: 3/10 (30) P: 0/10 (0) P: NE P: 1/8 (13) P: 53/53 (100) P: NE P: NE P: NE P: 3/6 (50)
M: 1/5 (20) M: NE P: NE M: 0/2 (0) M: 53/54 (98) M: NE M: NE M: NE M: NE
La Rosa et al73 CDX2-88 P: 0/7 (0) P: 0/6 (0) P: 3/7 (43) P: 5/24 (21) P: 12/12 (100) P: 5/5 (100) P: 2/2 (100) P: NE P: 0/14 (0)
M: NE M: 0/3 (0) M: 0/1 (0) M: NE M: 6/6 (100) M: NE M: 2/2 (100) M: NE M: 0/1 (1)
Saqi et al74 CDX2-88 P: 0/15 (0) P: 1/6 (17) P: 3/4 (75) P: 6/20 (30) P: 6/6 (100) P: 6/7 (86) P: NE P: NE P: 6/18 (33)
M: NE M: NE M: NE M: NE M: NE M: NE M: NE M: NE M: NE
Jaffee et al75 CDX2-88 P: 0/10 (0) P: 0/8 (0) P: NE P: NE P: 7/7 (100) P: 4/4 (100) P: NE P: NE P: 2/7 (29)
Volume 20, Number 5, September 2013

M: NE M: NE M: NE M: NE M: 4/4 (100) M: NE M: NE M: 1/1 (100) M: NE

Lin et al76 CDX2-88 P: 2/30 (7) P: 3/20 (15) P: NE P: 5/30 (17) P: 13/14 (93) P: 11/11 (100) P: NE P: 3/4 (75) P: 4/11 (36)
M: 0/2 (0) M: 0/1 (0) M: NE M: 0/1 (0) M: 5/5 (100) M: 2/2 (100) M: NE M: 2/2 (100) M: NE
Schmitt et al77 CDX2-88 P: 0/31 (0) P: 1/2 (50) P: 1/2 (50) P: 18/87 (21) P: 16/31 (52) P: 19/22 (86) P: NE P: 0/2 (0) P: 0/1 (0)
M: NE M: 1/1 (100) M: 2/2 (50) M: 1/18 (5) M: 11/18 (61) M: 3/4 (75) M: NE M: 1/2 (50) M: NE
Srivastava and Hornick78 CDX2-88 P: 0/20 (0) P: 0/5 (0) P: 0/5 (0) P: 3/31 (10) P: 21/21 (100) P: 11/11 (100) P: NE P: NE P: 0/12 (0)
M: NE M: NE M: NE M: 4/8 (50) M: 9/10 (90) M: NE M: NE M: NE M: NE
Hermann et al79 AMT28 P: NE P: NE P: 1/14 (7) P: 5/36 (16) P: NE P: NE P: NE P: NE P: NE
M: NE M: NE M: NE M: NE M: NE M: NE M: NE M: NE M: NE
Chan et al80 CDX2-88 P: 0/29 (0) P: NE P: NE P: 1/25 (4) P: 15/16 (94) P: 5/6 (83) P: 0/1 (0) P: 1/1 (100) P: 1/2 (50)
M: NE M: NE M: 0/2 (0) M: 2/5 (40) M: 2/2 (100) M: NE M: 2/2 (100) M: 1/1 (100) M: NE
Denby et al81 CDX2-88 P: NE P: NE P: NE P: NE P: NE P: NE P: NE P: NE P: NE
M: NE M: ND M: ND M: 6/16 (38) M: ND M: NE M: ND M: ND M: ND
Koo et al70 EPR2764Y P: 0/31 (0) P: 0/9 (0) P: NE P: 0/33 (0) P: 20/23 (87) P: NE P: NE P: NE P: 0/14 (0)
M: 0/5 (0) M: NE M: 0/1 (0) M: 1/28 (4) M: 33/37 (89) M: NE M: NE M: 1/1 (100) M: 0/1 (0)
Graham et al82 AMT28 P: NE P: NE P: 2/9 (22) P: 4/46 (9) P: NE P: NE P: NE P: 0/8 (0) P: 2/7 (29)
M: NE M: NE M: NE M: NE M: NE M: NE M: NE M: NE M: NE
Total P: 7/220 (3) P: 10/74 (14) P: 14/45 (31) P: 66/415 (16) P: 177/197 (90) P: 67/72 (93) P: 2/3 (67) P: 4/16 (25) P: 32/110 (29)
M: 1/13 (8) M: 2/6 (33) M: 3/7 (43) M: 15/84 (18) M: 129/142 (91) M: 5/6 (83) M: 4/4 (100) M: 6/7 (86) M: 0/2 (0)
F indicates functioning; M, metastatic; ND, not distinguishable; NE, not evaluated; NF, nonfunctioning; P, primary.

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Neuroendocrine Neoplasms: Assigning Site of Origin
Bellizzi
FIGURE 2. CDX2 expression in midgut well-differentiated neuroendocrine tumors (WDNET). A, Ileal tumor with predominantly type A
and focally type C histology. Note, also, the eosinophilic cytoplasmic granularity typical of enterochromaffin-cell tumors (hematoxylin
and eosin, 200). This patient had a concurrent ampullary adenocarcinoma. B, Two lymph nodes in the Whipple specimen contained
metastatic WDNET morphologically similar to that in the ileum. Diffuse, strong CDX2 expression suggests spread from the ileum
(immunoperoxidase, 200).
trabecular growth pattern did. Thus, in the ovary, CDX2 is
not helpful in distinguishing a primary insular WDNET
from a midgut metastasis; interestingly, these 2 tumor types
are essentially histologically identical. Also of note, tumors
in this study did not express TTF-1.
Thyroid Transcription Factor-1
Initially identified based on its ability to bind to the
thyroglobulin (TG) gene promoter,84 TTF-1 (also known as
NK2 homeobox 1) is the best known of several human NKX
homeodomain–containing transcription factors.85,86 It is
critical to lung, thyroid, and brain (diencephalon) develop-
ment, and it is expressed by type II pneumocytes, a
subpopulation of nonciliated bronchiolar epithelial cells,
follicular and parafollicular (C) cells, and neuroblasts in areas
of the developing diencephalon including the hypothalamus
and neurohypophysis.87–89 In addition to TG, TTF-1 pro-
motes the expression of thyroperoxidase, thyrotropin recep-
tor, surfactant proteins, and Clara cell secretory protein.
A mouse knockout is athyroid, lung development distal to
the lobar bronchi fails to take place, the ventral forebrain is
abnormally formed, and the pituitary is missing.90
TTF-1 is most commonly used in diagnostic pathology
as a marker of lung adenocarcinoma and thyroid
tumors.91–95 In the setting of a WDNET, TTF-1 has been
found to be a variably sensitive, although incredibly spe-
cific, marker of lung origin. Although unlikely to present as
a metastasis from an occult primary, expression is also
detected in nearly all medullary thyroid carcinomas, which
may additionally mark for calcitonin and carcinoembryonic
antigen.89,96–99 Nearly all studies of TTF-1 immunoex-
pression in NENs have used the same mouse MoAb
8G7G3/1 initially described by Holzinger et al100 in 1996,
which was produced against full-length recombinant rat
protein. A few more recent studies have also used the
mouse MoAb SPT24, raised against a 123 amino acid
sequence from the N-terminal region of human TTF-1. The
distinction is not inconsequential, with SPT24 generally
demonstrating increased sensitivity in tumor types in which
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Adv Anat Pathol  Volume 20, Number 5, September 2013
TTF-1 expression is typical, while some studies have also
demonstrated SPT24 positivity in occasional tumors in
which TTF-1 expression is considered unusual.101,102 For
example, Compérat et al101 found SPT24 and 8G7G3/1
positivity in 84% versus 65% of 86 lung adenocarcinomas
and 10% versus 0% of 41 lung metastases of colorectal
origin, respectively.
In 21 studies using the 8G7G3/1 clone, TTF-1
expression has been detected in 32% of 557 lung carcinoid
tumors (Table 5 and Figs. 3A–C). Including 1 additional
study in which a small number of TCs were examined with
a rabbit polyclonal antibody, rates of positivity in individ-
ual studies have ranged from 0% to 95%, with a median of
32%. ACs (mean 32%; median 40%) are possibly some-
what more likely to be positive than TCs (mean 28%;
median 21%). Spindle cell carcinoids frequently express
TTF-1 (mean and median 69%). Thirteen of 23 (57%)
metastases have been positive. In contrast, TTF-1 expres-
sion by a GEP WDNET is exceptional, reported to date in
0.6% of 708 primary tumors and 0% of 220 metastases. In
3 studies comparing SPT24 with 8G7G3/1, SPT24 was
more frequently expressed in lung carcinoids than was
8G7G3/1. For example, Matoso et al102 found 61%
(SPT24) versus 17% (8G7G3/1) of 23 TCs to be positive. In
1 study also evaluating GEP tumors, this increased sensi-
tivity was not at the expense of specificity, with La Rosa
et al112 not detecting SPT24 positivity in any (0%) of 103
tumors. The huge range of positivity (0% to 95%) cannot
be explained by differing antibody clones (it preceded the
first reports of SPT24 expression in this class of tumors),
use or nonuse of heat-induced epitope retrieval (use of
which is described in most of these studies), or differing
retrieval solutions (although a variety are described, citrate
has been most frequently used and has been associated with
both extremes of this range).
Paired Box Gene 8 (PAX8)
The transcription factor PAX8, 1 of 9 paired box
genes, is essential for thyroid development,115,116
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participates with PAX2 in kidney development,117,118 and
was found, through microarray-based gene expression
profiling, to be highly expressed in surface ovarian carci-
nomas.119 As such, detection of PAX8 expression has
emerged as an important diagnostic tool to identify carci-
nomas of the thyroid, Müllerian system, and kidney.120–122
As application of PAX8 IHC became more prevalent, a few
groups recognized strong expression in islets of Langerhans
and pancNETs and reported on the usefulness of PAX8
expression to suggest the pancreatic origin of a WDNET
(Table 6 and Figs. 4A, B).
In 7 studies using a commercially available poly-
clonal PAX8 antibody, expression has been detected in 145
of 209 (69%) primary and 46 of 84 (55%) metastatic pan-
cNETs. Although only a few duodenal (14 primaries) and
relatively few rectal (48 primaries) tumors have been stud-
ied, PAX8 expression (detected by the same antibody)
appears typical of these sites as well (79% and 58%,
respectively). PAX8 positivity has not been noted in 85
primary and 67 metastatic ileal tumors, and it has ranged
from 8% to 17% in tumors from the lung, stomach, and
appendix. Also of note, neither PAX8, PAX2, CDX2, nor
TTF-1 were found to be expressed in a series of 9 renal
WDNETs.135
Interestingly, up to 2010, the pancreatic developmental
biology literature was quiet on PAX8. Instead, PAX4 and
PAX6 had been shown critical to islet development, with
the PAX4 knockout mouse lacking mature b and d cells,136
the PAX6 knockout lacking a-cells,137,138 and the combined
PAX4/PAX6 knockout lacking any pancreatic neuro-
endocrine cells.137 The most frequently used PAX8 anti-
body is polyclonal, raised against an N-terminal peptide
that is highly conserved across the PAXs. It has been
demonstrated that PAX8 mRNA is expressed at very low
levels in human islets (relative to PAX6 and PAX4), that a
MoAb directed against the divergent C-terminal region
does not react with non-neoplastic islets or pancNETs, and
that the polyclonal PAX8 antibody cross-reacts with
PAX6.129
The authors of the above-referenced study suggest that
their results “cast doubts on the value of Pax8 as a pan-
creatic neuroendocrine tumor marker.”129 In a letter pub-
lished in the American Journal of Surgical Pathology, the
same group warns of the potential for “flawed diag-
nostics.”139 Another group reports on the development of a
PAX8 MoAb, which they suggest “offers a significant
advantage by simplifying interpretation and enabling a
more confident and accurate diagnosis.”130 In my opinion,
abandoning the polyclonal PAX8 antibody in this context
is akin to throwing out the proverbial baby with the
bathwater. As a GI pathologist, I embrace polyclonal car-
cinoembryonic antigen IHC’s cross-reactivity with biliary
glycoprotein to highlight the bile canaliculi diagnostic of
hepatocellular differentiation. Am I to divert my eyes from
granular cytoplasmic staining for TTF-1, suggesting the
possibility, again, of hepatocellular differentiation (another
cross-reactivity, this time with carbamoyl phosphate synthe-
tase I)? Even if polyclonal PAX8 does cross-react with PAX6,
the antibody remains an excellent marker of pancreatic (and
probably duodenal and rectal) WDNETs, better, in fact, than
PAX6 itself [in Lorenzo et al’s129 study, while polyclonal
PAX8 highlighted 7 of 9 (78%) pancNETs, a PAX6 MoAb
was positive in only 3 (33%)]. It can be argued that this cross-
reactivity adds value above and beyond that seen with the
recently developed PAX8 monoclonal.
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Neuroendocrine Neoplasms: Assigning Site of Origin
Insulin Gene Enhancer Binding Protein Isl-1
(Islet 1)
The homeodomain-containing transcription factor Islet 1
was initially isolated based on its ability to bind the insulin
gene enhancer.140 In the adult rat, Islet 1 expression was
detected in a, b, and d cells of the islets of Langerhans, cells in
the anterior and intermediate lobes of the pituitary, paraf-
ollicular (C) cells, chromaffin cells and some neurons in the
adrenal medulla, some sensory neurons in the dorsal root
ganglia, and subsets of motor neurons and central nervous
system nuclei involved in autonomic and endocrine control.141
In the developing embryo, Islet 1 is expressed in mesenchymal
cells of the dorsal pancreatic bud, as well as islet cells. An Islet
1 knockout mouse is characterized by failed exocrine devel-
opment from the dorsal but not the ventral pancreatic bud and
a complete absence of differentiated islet cells.142 Given its
importance in pancreatic, and in particular islet, development,
Islet 1 expression has been explored as a marker of pancNETs.
As with polyclonal PAX8, in addition to expression in this
group of tumors, Islet 1 positivity is also typical of duodenal
and rectal WDNETs (Table 6).
In a total of 5 studies investigating nearly 700
WDNETs, Islet 1 expression has been detected in 209 of 255
(82%) primary and 47 of 67 (70%) metastatic pancNETs.
Similarly, 92% of 25 duodenal and 86% of 22 rectal pri-
maries have been positive. Although only a handful of
metastases from the duodenum and rectum have been ana-
lyzed, the results are similar. Islet 1 expression is uncommon
to rare at other sites. Twelve percent (12%) of 94 pulmonary
and 17% of 42 appendiceal primaries have been positive,
whereas the few metastases studied have not. Islet 1 expres-
sion has not been described in a gastric tumor, and although
3% of 69 primary ileal tumors have been positive, all 68
metastases studied to date have been negative. A recent study
has highlighted frequent expression in medullary thyroid
carcinoma (9/9; 100%).131 In the same study, Islet 1 expres-
sion was also seen in most PDNECs including MCCs and
SCLCs, as well as neuroblastomas and paragangliomas/
pheochromocytomas. (Of note, this lack of “anatomic site
specificity” for a transcription factor in PDNECs has a
precedent; see directly below.) Compared with PAX8, Islet 1
boasts superior sensitivity for a pancreaticoduodenal or rectal
primary. Its principal disadvantage is that it is a “unitasker”
(ie, it has no other defined diagnostic application at this time).
KEY MARKERS IN DETERMINING THE SITE OF
ORIGIN OF A POORLY DIFFERENTIATED
NEUROENDOCRINE CARCINOMA
Thyroid Transcription Factor-1
Although most familiar as a marker of lung adeno-
carcinoma, the first reports of TTF-1 expression in human
tumors actually found more frequent expression in
pulmonary small cell carcinomas than in adenocarcinomas.
In their initial description of the 8G7G3/1 MoAb, Hol-
zinger et al100 found 5 of 6 (83%) SCLCs and 7 of 11 (64%)
adenocarcinomas to be positive, whereas Fabbro et al,103
using a rabbit polyclonal antibody, reported TTF-1
expression in 10 of 10 (100%) SCLCs and only 3 of 11
(27%) adenocarcinomas. TTF-1 is also expressed, although
much less frequently, by pulmonary LCNECs. Although
the reported rates have been highly variable, TTF-1 is also
expressed by at least a significant minority of extrapulmo-
nary PDNECs. However, expression by a cutaneous
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TABLE 5. TTF-1 Expression in Well-Differentiated Neuroendocrine Tumors

n (%)
References Antibody Pulmonary Tumors Gastroenteropancreatic Tumors
Fabbro et al103 Rabbit polyclonal TC 0/8 (0) NE
Folpe et al104 8G7G3/1 TC 18/51 (35) NE
AC 9/9 (100)
Agoff et al98 NR NE GE 0/49 (0)
P 0/15 (0)
Kaufmann and Dietel96 8G7G3/1 TC 4/9 (44) GEP 1/19 (5)
AC 2/3 (67)
Oliveira et al97 8G7G3/1 TC 16/17 (94) GE 0/26 (0)
AC 3/3 (100) P 0/10 (0)
MC 8/10 (80) Met GE 0/24 (0)
Cai et al105 8G7G3/1 C 11/16 (69) GE 1/46 (2)
MC 2/2 (100) P 0/12 (0)
Met GE 0/3 (0)
Met P 0/3 (0)
Sturm et al106 8G7G3/1 TC 0/27 (0) NE
AC 0/23 (0)
Zamecnik and Kodet107 8G7G3/1 TC 0/6 (0) NE
Chang et al108 8G7G3/1 TC 0/8 (0) NE
AC 0/3 (0)
Du et al109 8G7G3/1 TC 10/36 (28) GEP 0/17 (0)
AC 5/17 (29)
(SC) 12/16 (75)
Jerome Marson et al110 8G7G3/1 C 6/23 (26) NE
Pelosi et al4 8G7G3/1 TC 1/5 (25) NE
AC 0/2 (0)
Saqi et al74 8G7G3/1 TC 6/12 (50) GE 0/40 (0)
AC 2/3 (67) P 0/19 (0)
Hiroshima et al111 8G7G3/1 TC 0/7 (0) NE
AC 0/2 (0)
Lin et al76 8G7G3/1 TC 8/22 (36) GE 0/60 (0)
AC 5/8 (63) P 0/30 (0)
(SC) 8/13 (61) Met GE 0/10 (0)
MC 2/2 (100) Met P 0/1 (0)
Schmitt et al77 8G7G3/1 C 16/31 (52) GE 1/63 (2)
P 1/87 (1)
Met GE 0/18 (0)
Met P 0/28 (0)
Srivastava and Hornick78 8G7G3/1 C 7/20 (35) GE 0/54 (0)
P 0/31 (0)
Met GE 0/10 (0)
Met P 0/8 (0)
Matoso et al102 8G7G3/1 TC 4/23 (17) NE
SPT24 TC 14/23 (61)
La Rosa et al112 8G7G3/1 TC 4/41 (10) GE 0/60 (0)
AC 0/10 (0) P 0/26 (0)
(PC) 4/12 (33) Met GE 0/14 (0)
MC 0/4 (0) Met P 0/3 (0)
SPT24 TC 15/41 (37) GE 0/60 (0)
AC 2/10 (20) P 0/26 (0)
(PC) 9/12 (75) Met GE 0/14 (0)
MC 1/4 (25) Met P 0/3 (0)
Tsuta et al113 8G7G3/1 SC 9/13 (69) NE
Chan et al80 8G7G3/1 TC 11/24 (46) GE 0/26 (0)
AC 1/2 (50) P 0/25 (0)
Met GE 0/8 (0)
Met P 0/5 (0)
Denby et al81 8G7G3/1 NE Met GE 0/18 (0)
Met P 0/16 (0)
Koo et al70 8G7G3/1 C 16/31 (52) GE 0/46 (0)
MC 1/5 (20) P 0/33 (0)
Met GE 0/40 (0)
Met P 0/28 (0)
Masai et al114 8G7G3/1 C 2/50 (4) NE
SPT24 C 12/51 (24)
Range (median) (%) C 0-95 (32) GEP 0-5 (0)
TC 0-94 (21) GE 0-2 (0)

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TABLE 5. (continued)
n (%)
References Antibody Pulmonary Tumors Gastroenteropancreatic Tumors
AC 0-100 (40) P 0-1 (0)
SC 61-75 (69) Met GE 0 (0)
PC 33 (33) Met P 0 (0)
MC 0-100 (80)
Totals C 176/565 (31) GEP 4/708 (0.6)
TC 82/296 (28) GE 2/410 (0.5)
AC 27/85 (32) P 1/262 (0.4)
SC 29/42 (69) Met GE 0/131(0)
PC 4/12 (33) Met P 0/89 (0)
MC 13/23 (57)
AC indicates atypical carcinoid; C, primary carcinoid (encompassing typical and atypical); GE, gastroenteric (tubal gut); GEP, gastroenteropancreatic;
MC, metastatic carcinoid; Met, metastatic; NE, not evaluated; P, pancreatic; PC, peripheral carcinoid; SC, spindle cell carcinoid; TC, typical carcinoid.

PDNEC (ie, MCC) is exceptional. As such, TTF-1 IHC is
an essential component of the small panel of stains useful
in distinguishing visceral from cutaneous tumors, with
expression effectively ruling out the diagnosis of MCC.
Data from 57 studies examining TTF-1 expression in
PDNECs are summarized in Table 7. Expression has been
detected in 698 of 846 (83%) SCLCs and 101 of 282 (36%)
pulmonary LCNECs; the median reported rates for these 2
tumor types are 86% and 41%, respectively (Figs. 5A, B).
Two recent studies have compared the performance of the
8G7G3/1 and SPT24 clones, and, as with lung carcinoids,
positivity is more frequent with SPT24. For example, Masai
et al114 detected TTF-1 expression in 86% versus 68% of
SCLCs and 47% versus 23% of pulmonary LCNECs. TTF-
1 positivity has been reported in 200 of 550 (36%) extrap-
ulmonary PDNECs; rates in individual studies have ranged
from 0% to 83%, with a median of 33%. TTF-1 expression
has been reported in only 2 of 260 (0.8%) MCCs, each as a
single occurrence in 2 of 15 studies.
Cytokeratin 20
Using a combination of 2-dimensional gel electro-
phoresis, immunoblotting, and tryptic peptide mapping, in
1985 Moll and Franke203 identified a distinct cytoskeletal
protein of molecular weight 46,000 in 9 of 9 (100%) MCCs
and in none of 8 (0%) SCLCs. The protein colocalized with
one they had previously described in human intestinal cells,
GI tract adenocarcinomas, and a human colon cancer cell
line, which they had designated protein IT.204 Several years
later, protein IT was proven to be a keratin and designated
CK20.205 CK20 is normally expressed by gastric foveolar
epithelium, intestinal epithelium, urothelial umbrella cells,
and by Merkel cells in the skin. Diagnostically, it is gen-
erally used as a marker of intestinal adenocarcinomas, and
it is expressed by a smaller number of gastroesophageal;
pancreatobiliary; mucinous ovarian and lung; and urothe-
lial carcinomas.186,194 Germane to this discussion, CK20
expression is seen in the vast majority of MCCs, while
expression is unusual in SCLC. CK20 is similarly uncom-
mon in extrapulmonary PDNECs, with the exception of
those arising in the major salivary glands, which have been
described as “Merkel cell–like.”
CK20 expression has been noted in 416 of 472 (88%)
MCCs, with rates ranging from 67% to 100% in 24 studies
(median 91%) (Table 7). Expression is typically perinuclear/
dot-like but may also be diffuse (Fig. 6). CK20 positivity has
been reported in 18 of 373 (5%) SCLCs and 1 of 10 (10%)
pulmonary LCNECs. Eleven of these 18 were seen in 1 study
(11/33; 33%).145 Excluding this outlier study, the rate is 2%
(7/340); the median frequency in 19 studies is 0%. CK20
expression has been found in 19 of 30 (63%) major salivary
gland tumors and 20 of 331 (6%) nonmajor salivary gland
ones. In the largest series of major salivary gland small cell
carcinomas (SCNECs), CK20 expression was noted in 11 of

FIGURE 3. TTF-1 expression in bronchopulmonary carcinoid tumors. A, Liver metastasis from unknown primary composed of epi-
thelioid and spindled cells. Mitotic activity is inconspicuous (hematoxylin and eosin, 400). B, Ki-67 immunohistochemistry (IHC)
reveals a proliferation index of 4.2% (immunoperoxidase, 400). C, 8G7G3/1 IHC demonstrates modest, although fairly diffuse, TTF-1
expression, which in a WDNET suggests a bronchopulmonary origin; spindle cell carcinoids are more apt to be positive (immuno-
peroxidase, 600). This case was seen in consultation; the referring pathologist’s impression was metastatic small cell carcinoma.

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 TABLE 6. PAX8, Islet 1, and PDX1 Expression in Well-Differentiated Neuroendocrine Tumors
Bellizzi

n (%)
Jejunum/
References Antibody (Vendor) Lung Stomach Duodenum Pancreas Ileum Appendix Cecum Colon Rectum
Polyclonal PAX8
Long et al123 Polyclonal (Proteintech) P: 0/20 (0) P: 1/5 (20) P: 5/5 (100)
P: 42/63 (67) P: 0/31 (0) P: 4/19 (21) P: NE P: NE P: 11/13 (85)
M: NE M: NE M: NE M: 9/18 (50) M: 0/16 (0) M: NE M: NE M: NE M: NE
Sangoi et al124 Polyclonal (Proteintech) P: 0/21 (0) P: 2/20 (10) P: 6/8 (75)
P: 49/66 (74) P: 0/31 (0) P: 1/11 (9) P: NE P: NE P: 5/17 (29)
M: 0/2 (0) M: NE M: 0/2 (0)
M: 20/31 (65) M: 0/11 (0) M: NE M: NE M: NE M: 0/1 (0)
Ozcan et al125 Polyclonal (Proteintech) P: NE P: ND P: ND P: 6/17 (35) P: ND P: ND P: ND P: ND P: ND
M: NE M: ND M: ND M: ND M: ND M: ND M: ND M: ND M: ND
Haynes126 Polyclonal (Proteintech) P: 0/12 (0) P: NE P: 0/1 (0)
P: 12/18 (67) P: NE P: NE P: NE P: NE P: 0/1 (0)
M: 0/1 (0) M: NE M: 0/1 (0)
M: 2/7 (29) M: 0/3 (0) M: NE M: NE M: NE M: NE

296 | www.anatomicpathology.com
Laury et al127 Polyclonal (Proteintech) P: 0/2 P: NE P: NE P: NE P: NE P: NE P: NE P: NE P: NE
M: NE M: NE M: NE M: NE M: NE M: NE M: NE M: NE M: NE
Tacha128 Polyclonal (Biocare Medical) P: 0/7 P: NE P: NE P: 0/3 (0) P: NE P: NE P: NE P: NE P: 1/3 (33)
M: NE M: NE M: NE M: NE M: NE M: NE M: NE M: NE M: NE
Lorenzo et al129 Polyclonal (Proteintech) P: NE P: NE P: NE P: 7/9 (78) P: NE P: NE P: NE P: NE P: NE
M: NE M: NE M: NE M: NE M: NE M: NE M: NE M: NE M: NE
ab53490 (Abcam) P: 0/9 (0)
M: NE
Koo et al70 Polyclonal (Proteintech) P: 7/31 (23) P: 2/9 (22) P: NE P: 29/33 (88) P: 0/23 (0) P: NE P: NE P: NE P: 11/14 (79)
M: 0/5 (5) M: NE M: 0/1 (0) M: 15/28 (54) M: 0/37 (0) M: NE M: NE M: 0/1 (0) M: 0/1 (0)
Tacha et al130 BC12 (Biocare Medical) P: 0/16 (0) P: 0/3 (0) P: ND P: NE P: ND P: 0/2 (0) P: ND P: ND P: 0/2 (0)
M: NE M: NE M: NE P: NE M: NE M: NE M: NE M: NE M: NE
Total Polyclonal P: 7/93 (8) P: 5/34 (15) P: 11/14 (79) P: 145/209 (69) P: 0/85 (0) P: 5/30 (17) P: ND P: NE P: 28/48 (58)
M: 0/8 (0) M: ND M: 0/4 (0) M: 46/84 (55) M: 0/67 (0) M: ND M: ND M: 0/1 (0) M: 0/2 (0)
Monoclonal P: 0/16 (0) P: 0/3 (0) P: ND P: 0/9 (0) P: ND P: 0/2 (0) P: ND P: ND P: 0/2 (0)
M: NE M: NE M: ND M: NE M: NE M: NE M: NE M: NE M: NE
Islet 1
Schmitt et al77 40.3A4 P: 5/31 (16) P: 0/2 (0) P: 1/2 (50) P: 58/84 (69) P: 1/30 (3) P: 4/20 (20) P: NE P: 0/2 (0) P: 0/1 (0)
(DSHB) M: NE M: 0/1 M: 2/2 (100) M: 12/18 (67) M: 0/17 (0) M: 0/4 (0) M: NE M: 0/2 (0) M: NE
Adv Anat Pathol

Hermann et al79 C-terminal portion P: NE P: NE P: 14/14 (100) P: 32/36 (89) P: NE P: NE P: NE P: NE P: NE



(DSHB) M: NE M: NE M: NE M: NE M: NE M: NE M: NE M: NE M: NE

r
Koo et al70 40.3A4 (DSHB) P: 2/31 (6) P: 0/9 (0) P: NE P: 27/33 (82) P: 0/23 (0) P: NE P: NE P: NE P: 12/14 (86)
M: 0/5 (0) M: NE M: 0/1 (0) M: 19/28 (68) M: 0/37 (0) M: NE M: NE M: 0/1 (0) M: 1/1 (100)
Graham et al82 1H9 (Abcam) P: 0/17 (0) P: 0/10 (0) P: 8/9 (89) P: 92/102 (90) P: 1/16 (6) P: 3/22 (14) P: NE P: 3/8 (38) P: 7/7 (100)
M: 0/2 (0) M: NE M: NE M: 16/21 (76) M: 0/14 (0) M: NE M: 0/1 (0) M: NE M: 2/2 (100)
Agaimy et al131 1H9 (Abcam) P: 4/15 (27) P: NE P: NE P: NE P: NE P: NE P: NE P: NE P: NE
M: NE M: NE M: NE M: NE M: NE M: NE M: NE M: NE M: NE
Total P: 11/94 (12) P: 0/21 (0) P: 23/25 (92) P: 209/255 (82) P: 2/69 (3) P: 7/42 (17) P: NE P: 3/10 (30) P: 19/22 (86)
M: 0/7 (0) M: 0/1 (0) M: 2/3 (67) M: 47/67 (70) M: 0/68 (0) M: 0/4 (0) M: 0/1 (0) M: 0/3 (0) M: 3/3 (100)
PDX1
Srivastava and Hornick78 Polyclonal (Santa Cruz) P: 0/20 (0) P: 3/5 (60) P: 4/5 (80) P/M: 11/39 (28)* P: 0/21 (0) P: 6/11 (55) P: NE P: NE P: 2/12 (17)
M: NE M: NE M: NE M: 0/10 (0) M: NE M: NE M: NE M: NE
Fendrichet al132w Polyclonal (Chemicon) P: NE P: NE P: 0/15 (0) P: 8/8 (100) P: NE P: NE P: NE P: NE P: NE
M: NE M: NE M: 0/3 (0) M: 5/5 (100) M: NE M: NE M: NE M: NE M: NE
Zhang et al133 Polyclonal (Abcam) P: NE P: NE P: NE P: 47/97 (48) P: NE P: NE P: NE P: NE P: NE
M: NE M: NE M: NE M: NE M: NE M: NE M: NE M: NE M: NE

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Volume 20, Number 5, September 2013
Adv Anat Pathol  Volume 20, Number 5, September 2013 Neuroendocrine Neoplasms: Assigning Site of Origin

15 (73%) tumors; TTF-1 expression was restricted to 3 of the

P: 2/14 (14)
4 CK20-negative cases.157
P: 0/2 (0)
M: NE

M: NE

M: NE
P: NE

P: NE
P: NE

OTHER MARKERS OF INTEREST IN WELL-

DIFFERENTIATED NEUROENDOCRINE TUMORS
M: 0/1 (0)

M: 0/1 (0)
P: 0/1 (0)

P: 0/1 (0)

Cytokeratins 7 and 20
M: NE
P: NE

P: NE
P: NE

A good deal of effort has been put forth to define the

expression of various keratin types in carcinomas from
diverse anatomic sites, with the goal of applying these data
M: 0/2 (0)

M: 0/2 (0)
P: 0/1 (0)

P: 0/1 (0)

to questions of tumor type (eg, adenocarcinoma vs. squ-

M: NE
P: NE

P: NE
P: NE

amous cell carcinoma) and, especially, site of origin. I have

seen pathologists attempt to extrapolate the findings from
what principally has been the study of non-neuroendocrine
P: 7/17 (41)
P: 1/6 (17)

carcinomas to WDNETs. In fact, there is actually a relative

M: NE

M: NE

M: NE

M: NE

paucity of data on the expression of individual keratins in

P: NE

P: NE

these tumors, with CK7 and CK20, given their wide clinical
application, having received the most attention (there is a
separate literature on the role of CK19 as a prognostic
marker in pancNETs). Although only up to a few hundred
M: 0/12 (0)
P: 0/16 (0)

P: 0/37 (0)
M: 0/2 (0)

tumors have been studied, the results have been fairly

M: NE

M: NE
P: NE

P: NE

consistent, such that conclusions can be drawn (Table 8).

CK7 is frequently, although not invariably, expressed
by carcinoid tumors of the lung. In 5 studies including a
total of 87 tumors, frequency of expression in individual
P: 114/210 (54)
M: 10/10 (100)
P: 18/36 (50)

P: 23/44 (52)

P: 18/25 (72)

studies has ranged from 4% to 66% (median 30%); overall,

M: 5/5 (100)

40% of pulmonary WDNETs have expressed CK7.

M: NE

M: NE

Expression is less frequent in both tubal gut (11/131; 8%)

and pancNETs (8/37; 22%), with the caveat that relatively
few pancreatic tumors have been examined. Although CK7
expression is more common in lung tumors, given more
P: 12/14 (86)

P: 16/34 (56)
M: 2/2 (100)

M: 2/5 (40)

frequent metastasis from ileal and pancreatic primaries,

CK7 positivity at a metastatic site probably would be most
M: NE

M: NE
P: NE

P: NE

often seen with spread from the pancreas. CK7 IHC is

neither sensitive nor specific enough to be useful in this
diagnostic application.
P: 3/5 (60)

CK20 is expressed by around a quarter of tubal gut

M: NE

M: NE

M: NE

M: NE
P: NE

P: NE

P: 3/29 (10) P: NE

WDNETs, perhaps slightly less often in pancNETs, and

almost never in carcinoid tumors of the lung. In 7 studies
M indicates metastatic; ND, not distinguishable; NE, not evaluated; P, primary.

including a total of 114 tubal gut tumors, frequency of

expression in individual studies has ranged from 15% to
P: 3/49 (6)

100% (median 24%); overall, expression has been noted in

M: NE

M: NE

M: NE

M: NE
P: NE

P: NE

24%. Of note, the 2 earliest studies describing CK20 pos-

itivity in 100% and 86% of 2 and 7 tumors, respectively,
reported expression in rare cells only.184,187 For pancNETs,
frequency of expression in 4 studies has ranged from 12%
to 33% (median 17%); in total, 9 of 49 (18%) tumors have
*Primary tumors and metastases were not distinguished.
Polyclonal (Santa Cruz)
267712 (R&D Systems)

267712 (R&D Systems)

been CK20 positive. Chan et al80 recently reported CK20

expression in 1 of 29 (3%) bronchopulmonary carcinoids; 56
tumors in 4 previous studies had all been negative. Although
wStudy restricted to (functioning) gastrinomas.

relatively insensitive as a marker of GEP WDNETs, CK20

expression argues against a pulmonary origin.
Neuroendocrine Secretory Protein 55
Neuroendocrine secretory protein 55 (NESP55) is 1 of 5
main transcripts of the GNAS locus. It is a member of the
granin family (like the more familiar chromogranin) and is
found in dense-core secretory granules in the adrenal medulla,
pituitary, and several parts of the brain.209 A few studies have
Hermann et al79

suggested that NESP55 expression in a WDNET is in keeping

Park et al134

Chan et al80

with a pancreatic origin. Jakobsen et al210 found NESP55

expression in 14 of 25 (56%) pancNETs and 0 of 15 (0%) ileal
Total

WDNETs. NESP55 expression was also detected in non-

neoplastic islets, most frequently in b cells. Also of note, 19 of
19 (100%) pheochromocytomas were positive. Similarly,

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Bellizzi
FIGURE 4. Polyclonal PAX8 expression in pancreatic neuroendocrine tumors. A, Interface of a primary pancreatic tumor with trabecular
architecture and non-neoplastic pancreas (hematoxylin and eosin, 200). B, Polyclonal PAX8 immunostain highlights both the tumor
and adjacent non-neoplastic islets of Langerhans (immunoperoxidase,  200).
Srivastava et al211 described NESP55 expression in 14 of 19
(74%) pancNETs, 10 of 10 (100%) pheochromocytomas, and
0 of 11 (0%) ileal WDNETs. In addition, there was no staining
in 4 gastric tumors and focal staining (< 5% of cells) in 1 of 4
rectal (25%) and 1 of 15 (7%) lung tumors. Srivastava and
Hornick subsequently extended this finding in a larger group
of tumors, reporting expression in 16 of 39 (41%) pancNETs
and no expression in gastric (0/5), duodenal (0/5), ileal (0/31),
or appendiceal tumors (0/11). As in the prior study, expression
was noted in a rare rectal (1 of 12; 8%) and lung (1 of 20; 5%)
tumor.78 Recently, Denby et al81 reported NESP55 expression
in 5 of 16 (31%) and 0 of 16 (0%) metastatic pancreatic and
tubal gut WDNETs, respectively.
Of note, all 4 of these studies made use of a polyclonal
rabbit antibody gifted by the same Austrian investigator. An
internet search identified several commercial sources of anti-
NESP55, including at least 1 claiming to have been tested in
IHC of formalin-fixed, paraffin-embedded tissue. Although
the specificity of these commercial antibodies for pancNETs
would have to be verified, NESP55 represents an attractive
option in this setting. A drawback of this marker is its rela-
tively limited clinical utility beyond this specific application.
Pancreatic and Duodenal Homeobox 1
Pancreatic and duodenal homeobox 1 (PDX1) is
another homeodomain-containing transcription factor, ini-
tially described based on its ability to bind to and trans-
activate the insulin and somatostatin gene promoters.212,213 In
the developing embryo, PDX1 is expressed in the dorsal and
ventral pancreatic buds and the intervening duodenum; in the
pancreas, expression is gradually extinguished in acinar and
ductal epithelium, such that, by around birth, PDX1 is rel-
atively islet-restricted.214 PDX1 knockout mice demonstrate
severe pancreatic hypoplasia, duodenal malformation,
absence of Brunner glands, and decreased numbers of duo-
denal enteroendocrine cells.214,215 In the adult, PDX1
expression has been noted in islets of Langerhans, cen-
troacinar cells, rare intralobular and interlobular ductal cells,
and duodenal epithelium, and not in pancreatic acinar
parenchyma.134 Given its importance in pancreaticoduodenal
development and its continued expression in islets and the
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Adv Anat Pathol  Volume 20, Number 5, September 2013
duodenum, PDX1 has been explored as a marker of
WDNETs of these organs (Table 6).
There are very little data regarding PDX1 expression
outside of the pancreas—where it has been demonstrated in
114 of 220 (54%) primary tumors and 10 of 10 (100%)
metastases (data from 1 study are excluded from these
totals because primaries and metastases were not reported
separately). Although in 3 studies PDX1 was expressed by
most duodenal tumors [16/19 (84%) primaries and 2/2
(100%) metastasis],78–80 in a single study that only looked
at functioning gastrinomas, PDX1 was not expressed at all
(15 primaries and 3 metastases).132 Hermann et al79 ach-
ieved a very different result, with PDX1 expression noted in
10 of 10 (100%) gastrin-expressing duodenal tumors [5 of
these patients had Zollinger-Ellison syndrome (ZES)].
PDX1 expression appears to be relatively uncommon in
lung primaries (3/49; 6%) and, importantly, has not been
observed in 37 jejunoileal primaries and 12 metastases
(0%). There are almost no data from the stomach (3/5
primaries; 60%) and little data from the appendix (7/17
primaries; 41%) or rectum (2/14 primaries; 14%). PDX1
may have some role as a marker of pancreaticoduodenal
WDNETs. It may be less frequently positive in rectal
tumors than the other similar markers PAX8 and Islet 1,
although additional data would be welcomed.
Peptide Hormones
Commercial antibodies to a wide variety of peptide
hormones are available. A partial list of those normally
expressed in the GEP system is provided in Table 9. Aside
from gastrin, which is often used in gastric mucosal biopsies
as part of the workup of a suspected case of autoimmune
atrophic gastritis, these are generally considered of limited
clinical utility. As such, most laboratories will only carry a
few of these. Of note, functional syndromes are defined
based on the presence of characteristic symptoms, rather
than predominant or even exclusive immunohistochemical
expression of a peptide hormone. Numerous studies have
demonstrated that WDNETs, regardless of site of origin,
tend to express >1 hormone.14,216–222 Furthermore, meta-
stases may produce fewer or even additional peptides than
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TABLE 7. TTF-1 and CK20 Expression in Poorly Differentiated TABLE 7. (continued)

Neuroendocrine Carcinomas
n (%)
n (%)
Extrapulmonary
Extrapulmonary References Pulmonary Merkel Cell [Site]
References Pulmonary Merkel Cell [Site]
Hiroshima SC: 13/23 (57) NE NE
TTF-1 et al111 LC: 4/17 (24)
Holzinger SC: 5/6 (83) NE NE Yun et al169 NE NE SC: 15/21 (71)
et al100 [esophagus]
Fabbro SC: 10/10 NE NE Carlson SC: 20/22 (91) NE SC: 0/7 (0)
et al103 (100) et al170 [ovary,
Di Loreto SC: 38/41 (93) NE NE hypercalcemic-
et al143 type]*
Folpe et al104 SC: 20/21 (95) NE NE SC: 1/2 (50)
LC: 6/8 (75) [ovary, lung-
Byrd-Gloster SC: 35/36 (97) 0/21 (0) NE type]
et al144 SC: 3/8 (38)
Hanly et al145 SC: 28/33 (85) 0/21 (0) NE [uterine cervix]
Agoff et al98 NE 0/6 (0) SC: 7/16 (44) El Demellawy NE NE SC: 0/10 (0)
Kaufmann SC: 30/37 (81) 0/16 (0) SC: 12/15 (80) et al171 [colon]
and Dietel96 LC: 2/4 (50) LC: 1/4 (25) Alijo Serrano NE NE SC: 11/44 (25)
Ordóñez146 SC: 27/28 (96) 0/18 (0) SC: 4/36 (11) et al172 LC: 2/2 (100)
Cheuk et al147 SC: 43/52 (83) 0/23 (0) SC: 21/50 (42) [bladder]
Shin et al148 NE NE SC: 2/10 (20) Lin et al76 SC: 28/30 (93) 0/3 (0) NE
[breast] Kargi et al173 SC: 28/28 NE NE
Leech et al149 SC: 10/10 0/11 (0) NE (100)
(100) Lu et al174 NE NE SC: 9/15 (60)
Sturm et al150 LC: 18/44 (41) NE NE [esophagus]
Chhieng SC: 2/5 (40) NE NE Wang and NE NE SC: 23/44 (52)
et al151 Epstein175 [prostate]
Zamecnik and SC: 8/9 (89) NE NE Ralston SC: 43/59 (73) 1/30 (3) NE
Kodet107 et al176
Sturm et al106 SC: 47/55 (85) NE NE McCluggage NE NE SC: 11/13 (85)
LC: 31/64 (48) et al177 LC: 4/8 (50)
Wu et al152 SC: 20/23 (87) NE NE [uterine cervix]
Myong153 SC: 14/16 (88) NE NE Li et al178 NE NE SC: 9/42 (21)
Yamamoto NE NE SC: 2/6 (33) [tubal gut]
154
et al [esophagus] Lewis et al179 NE 0/12 (0) HG: 5/14 (36)
Yang et al155 SC: 9/9 (100) 0/22 (0) NE [head and neck
Liu156 SC: 6/8 (75) NE NE mucosa]
Chang et al108 SC: 19/36 (53) NE NE La Rosa SC: 14/19 (73) 0/4 (0) HG: 5/41 (12)
157
Nagao et al NE NE SC: 3/15 (20) et al112 LC: 4/13 (31) [clone [clone 8G7G3/
[major salivary [clone 8G7G3/1] 1]
gland] 8G7G3/1]
Du et al109 LC: 6/16 (38) NE LC: 0/3 (0) SC: 16/19 (84) 0/4(0) HG: 8/41 (20)
Jerome SC: 24/30 (80) NE NE LC: 10/13 (77) [clone [clone SPT24]
Marson LC: 6/10 (60) [clone SPT24]
et al110 SPT24]
Soriano NE NE SC: 2/10 (20) Sidiropoulos SC: 23/30 (77) 1/40 (3) NE
et al158 [bladder] et al180
McCluggage NE NE SC: 0/15 [ovary, Fernández- NE NE SC: 1/6 (16)
et al159 hypercalcemic- Aceñero [bladder]
type]* et al181
Liu and SC: 6/6 (100) NE NE Thompson NE NE SC: 0/7 (0)
Farhood160 et al182 [bladder]
Zhang et al161 SC: 26/28 (93) NE NE Quinn et al183 NE NE SC: 9/25 (36)
Pelosi et al4 SC: 6/9 (67) NE NE LC: 0/2 (0)
Nassar et al162 NE NE HG: 0/9 (0) Masai et al114 SC: 46/68 (68) NE NE
[ampulla] LC: 24/106
Llombart NE 0/20 (0) NE (23) [clone
et al163 8G7G3/1]
Jones et al164 NE NE SC: 17/44 (39) SC: 59/69 (86)
[bladder] LC: 50/106
Wu et al165 SC: 15/16 (94) NE NE (47) [clone
Bobos et al166 SC: 11/13 (85) 0/13 (0) SPT24]
Yao et al167 NE NE SC: 15/18 (83) Denby et al81 SC: 12/17 (71) NE SC: 6/10 (60)
[prostate] [metastatic] [metastatic]
Kalhor SC: 12/13 (96) NE NE
et al168

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Bellizzi Adv Anat Pathol  Volume 20, Number 5, September 2013

TABLE 7. (continued) TABLE 7. (continued)

n (%) n (%)
Extrapulmonary Extrapulmonary
References Pulmonary Merkel Cell [Site] References Pulmonary Merkel Cell [Site]
Range SC: 40-100 0-3 (0) 0-83 (33) Ishida et al199 NE NE SC: 0/10 (0)
(median) (86) [uterine cervix]
(%) LC: 23-75 (41) Nassar et al162 NE NE HG: 3/8 (38)
Total SC: 698/846 2/260 (0.8) 200/550 (36) [ampulla]
(83) Llombart NE 15/20 (75) NE
LC: 101/282 et al163
(36) Jones et al164 NE NE SC: 0/44 (0)
Using 8G7G3/1 [bladder]
clone data Acebo et al200 NE 8/9 (89) NE
from Masai Bobos et al166 SC: 1/13 (8) 13/13 (100) NE
and La Yao et al167 NE NE SC: 2/18 (11)
Rosa [prostate]
CK20 El Demellawy NE NE SC: 2/10 (20)
Moll et al184 SC: 3/15 (20) 15/15 (100) NE et al171 [colon]
[rare cells] Alijo Serrano NE NE SC: 3/44 (7)
Brinkschmidt NE 17/18 (94) NE et al172 LC: 0/2 (0)
et al185 [bladder]
Wang et al186 SC: 0/11 (0) NE NE Saeb-Lima NE 7/7 (100) NE
Miettinen187 SC: 0/6 (0) 5/5 (100) NE et al201
Chan et al188 SC: 1/37 (3) 33/34 (97) SC: 4/52 (8) McCluggage NE NE SC: 3/13 (23)
[overall] et al177 LC: 1/8 (13)
including 3/5 (60) [uterine cervix]
[major salivary Lewis et al179 NE 11/12 (92) HG: 3/14 (21)
gland] [head and neck
Schmidt SC: 0/18 (0) 43/56 (77) NE mucosa]
et al189 Sidiropoulos SC: 0/30 (0) 35/40 (88) NE
Metz et al190 SC: 0/22 (0) 6/6 (100) NE et al180
Scott and SC: 0/6 (0) 9/10 (90) NE Fernández- NE NE SC: 0/6 (0)
Helm191 Aceñero LC: NE [bladder]
Byrd-Gloster SC: 1/36 (3) 16/21 (76) NE et al181
et al144 Chernock NE NE SC: 4/5 (80)
Hanly et al145 SC: 11/33 (33) 20/21 (95) NE et al202 LC: 1/2 (50)
Nicholson SC: 0/5 (0) 18/27 (67) NE [parotid]
et al192 Range 0-33 (0) 67-100 (91) 0-38 (3)
Ordóñez146 SC: 1/28 (4) 16/18 (89) SC: 0/36 (0) (median) [nonmajor
[overall] (%) salivary gland]
including 0/2 (0) 0-73 (60) [major
[salivary gland] salivary gland]
Shin et al193 NE NE SC: 0/9 (0)[breast] Total 19/383 (5) 416/472 (88) 20/331 (6)
Chu et al194 SC: 0/7 (0) 7/9 (78) NE [nonmajor
Jensen et al195 NE 23/26 (88) NE salivary gland]
Cheuk et al147 SC: 0/52 (0) 23/23 (100) SC: 2/50 (4) 19/30 (63) [major
[overall] salivary gland]
including 0/1 (0)
[parotid] *Data for small cell carcinoma of the ovary, hypercalcemic-type not
Leech et al149 SC: 0/10 (0) 10/11 (91) NE included in the total as this tumor type of uncertain histogenesis is non-
neuroendocrine.
Chhieng SC: 0/5 (0) NE NE HG indicates high-grade neuroendocrine carcinoma (ie, small cell and
et al151 large cell neuroendocrine carcinomas not distinguished); LC, large cell
Kende et al 196 NE NE SC: 0/2 (0) neuroendocrine carcinoma; NE, not evaluated; SC, small cell neuro-
LC: 0/2 (0) endocrine carcinoma.
M: 0/1 (0) [tubal
gut]
Mott et al197 NE 16/18 (89) NE
Yang et al155 SC: 0/9 (0) 19/22 (86) NE
Nagao et al157 NE NE SC: 11/15 (73)
matched primary tumors.219 I have used peptide hormone
[major salivary IHC in the workup of a WDNET in 3 contexts: (1) to
gland] distinguish pancreatic neuroendocrine (pseudo)hyperplasia,
Jerome SC: 0/30 (0) NE NE which should express some combination of insulin, gluca-
Marson LC: 1/10 (10) gon, somatostatin, and pancreatic polypeptide, from a
et al110 microadenoma (defined as a nonfunctioning pancNET
Soriano NE NE SC: 0/10 (0) <0.5 cm), which tends to express a single hormone; (2) in
et al158 [bladder] operated patients with multiple simultaneous tumors and a
Tanaka NE 31/31 (100) NE functional syndrome (MEN1-ZES), to help determine, for
et al198
example, which of the resected tumors (if any) is the source
of the syndrome; (3) in patients with metachronous tumors,

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Adv Anat Pathol  Volume 20, Number 5, September 2013 Neuroendocrine Neoplasms: Assigning Site of Origin

FIGURE 5. TTF-1 expression in small cell carcinoma (SCNEC). A, Metastatic SCNEC to the liver (hematoxylin and eosin,  400). B, The
tumor expresses TTF-1. Note, also, cross-reactivity with carbamoyl phosphate synthetase I, accounting for the granular, cytoplasmic
staining in hepatocytes at the lower right (immunoperoxidase, 400). TTF-1 expression is typical of small cell lung carcinoma (B85%)
but is seen often enough in extrapulmonary SCNEC (B35%) that it is not useful in assigning the site of origin; Merkel cell carcinoma,
however, is negative.

in which the first was known to express a dominant hor- of 96 (58%) pancNETs, 0 of 29 (0%) tubal gut WDNETs,
mone (Figs. 7A–D). and 1 of 15 (7%) lung TCs.224 Tempering enthusiasm
for the marker, expression was much more frequent in
Progesterone Receptor benign tumors (72%) than in malignant ones (31%; defined
Progesterone receptor IHC is a standard-of-care pre- in this study by the presence of local invasion or metastasis),
dictive marker in breast cancer and is widely used as part of a
panel to help determine the site of origin of a carcinoma of
unknown primary. In the setting of a WDNET, PR
expression has been proposed as indicative of pancreatic TABLE 8. CK7 and CK20 Expression in Well-Differentiated
origin. Doglioni et al223 reported PR expression in 40% to Neuroendocrine Tumors
75% of non-neoplastic islet cells, especially prevalent n (%)
in a cells, and 7 of 18 (39%) pancNETs. Estrogen receptor
(ER) expression was not detected. In a follow-up study References Lung Gastrointestinal Pancreatic
from the same group, PR expression was detected in 56 CK7
Broers et al206 3/10 (30) NE NE
Chu et al194 2/9 (22) 2/15 (13) NE
Cai et al105 10/16 5/46 (11) 6/12 (50)
(63)
Jerome Marson et al110 1/23 (4) NE NE
Alsaad et al207 NE 0/25 (0)* NE
Jiang et al208 NE 2/19 (11)* NE
Chan et al80 19/29 2/26 (8) 2/25 (8)
(66)
Range (median) 4-66 (30) 0-13 (11) 8-50 (29)
Total 35/87 11/131 (8) 8/37 (22)
(40)
CK20
Moll et al184 NE 2/2 (100)w 1/5 (20)
Miettinen187 0/8 (0) 6/7 (86)w 1/7 (14)
Chu et al194 0/9 (0) 1/15 (7) NE
Cai et al105 0/16 (0) 11/46 (24) 4/12 (33)
Jerome Marson et al110 0/23 (0) NE NE
Alsaad et al207 NE 4/25 (16)* NE
Jiang et al208 NE 6/19 (32)* NE
Chan et al80 1/29 (3) 4/26 (15) 3/25 (12)
Range (median) 0-1 (0) 15-100 (24) 12-33 (17)
Total 1/85 (1) 30/114 (24) 9/49 (18)
FIGURE 6. CK20 expression is seen in B90% of Merkel cell *Appendix only.
carcinomas; expression is typically perinuclear/dot-like (immuno- wOnly rare cells positive.
peroxidase, 600). In contrast, CK20 expression by visceral NE indicates not evaluated.
SCNECs (with the exception of parotid tumors) is unusual.

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Bellizzi Adv Anat Pathol  Volume 20, Number 5, September 2013

identified. Zhao et al225 found (focal) PR and ER expres-

TABLE 9. Commercially Available Antibodies to Peptide sion in 1 of 42 (2%) ovarian carcinoid tumors. Sica et al226
Hormones
studied PR and ER expression in pulmonary NENs and
Gastrin found more frequent PR expression than did the Milanese
Insulin group: 11 of 42 (26%) TCs and 2 of 7 (29%) ACs. In
Glucagon addition, ER expression was seen in over half the carci-
Somatostatin
noids. Given the wide availability of PR IHC and its
Pancreatic polypeptide
Gastric inhibitory peptide
potential application as a secondary marker in assigning the
Motilin site of origin in a WDNET, PR expression in these tumors
Secretin warrants additional investigation.
Cholecystokinin
Vasoactive intestinal polypeptide
Glicentin Prostatic Acid Phosphatase
Peptide YY
Prostatic acid phosphatase (PrAP) is often used along
with prostate-specific antigen to support a diagnosis of
and expression was detected in only 1 of 7 (14%) liver prostate cancer, either in the workup of a carcinoma of
metastases. unknown primary or in the setting of a high-grade tumor in
This finding has apparently received relatively little the prostate or bladder for which urothelial carcinoma also
attention, as only 2 other relevant published studies were enters the differential diagnosis. The frequent expression of

FIGURE 7. Peptide hormone immunohistochemistry. A, A 60-year-old woman with symptomatic hypoglycemia was found to have a
1.2 cm pancreatic tumor, which is composed of polygonal cells exhibiting a trabecular growth pattern and with prominent peliosis
(hematoxylin and eosin, 400). B, The tumor expresses insulin (immunoperoxidase,  400). C, Preoperative staging workup also
demonstrated a small lung tumor, which was subsequently resected. It is composed of somewhat fusiform cells, exhibiting trabecular to
pseudoglandular growth (hematoxylin and eosin, 400). D, This tumor does not express insulin (immunoperoxidase,  400). Given
subtly distinct morphologies and disparate insulin expression, these tumors were interpreted as separate primaries. Of note, insulinomas
are rarely metastatic at presentation.

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Adv Anat Pathol  Volume 20, Number 5, September 2013 Neuroendocrine Neoplasms: Assigning Site of Origin

FIGURE 8. S-100 immunohistochemistry. A, Metastasis from the appendiceal tumor depicted in Figure 1D. Note the nested growth and
eosinophilic cytoplasmic granularity typical of midgut well-differentiated neuroendocrine tumors (hematoxylin and eosin, 200).
B, S-100 immunohistochemistry performed on the metastatic focus highlights sustentacular cells (immunoperoxidase,  400). These
were also noted in the primary tumor. The presence of sustentacular cells has been suggested as useful in distinguishing appendiceal
from ileal tumors, although there are little to no data in the metastatic setting.

PrAP by rectal WDNETs (leading to an incorrect diagnosis
of prostate cancer in a small, crushed rectal biopsy) repre-
sents a “classic pitfall” in diagnostic IHC. In the AFIP
series of 81 rectal (and 3 sigmoid) WDNETs, PrAP
expression was detected in 82%.222
PrAP expression is not restricted to rectal tumors,
although at most other sites it appears more infrequent. In
the AFIP series of 51 jejunoileal WDNETs, PrAP was
expressed in 10 (20%).10 In an earlier study, the same group
reported expression in 15% of nonrectal WDNETs (includ-
ing gastric, small intestinal, and appendiceal tumors).227
Azumi et al228 found even less frequent expression in 28
foregut-derived and midgut-derived tumors (focal, weak
expression in 1 lung tumor). In contrast, frequent expression
has been reported in insulinomas229,230 and strumal carci-
noids,231–233 although this finding has not been well vetted in
these tumor types. Again, given wide availability and appa-
rent differential expression across sites of origin, PrAP may
have some use as a secondary marker in this context.
recently, using the more specific marker SOX10, Tsuta
et al237 detected sustentacular cells in 51 of 78 (65%)
pulmonary carcinoids (more frequently in spindle cell/
peripheral tumors) and 0 of 35 (0%) pulmonary PDNECs.
One important study casts doubt on the appendiceal spe-
cificity of sustentacular cells in midgut WDNETs, with
Al-Khafaji et al238 finding S-100-positive sustentacular cells
in 3 of the 11 (27%) jejunoileal WDNETs they studied.
I recently encountered an appendiceal WDNET
metastatic to the uterus, in which sustentacular cells were
demonstrable in both the primary and metastatic tumor
(Figs. 8A, B). That sustentacular cells were present in the
metastasis is intriguing, and the possibility that S-100 IHC
might have some usefulness in assigning the site of origin of
a WDNET is considered, although, obviously an appendi-
ceal tumor presenting as a metastatic WDNET of unknown
primary would be very unusual. S-100 could also be
potentially useful in distinguishing a tumor arising at the
base of the appendix from an ileocecal one.

S-100 Somatostatin Receptor 2A

The detection of S-100 protein expression, including as
a marker of melanocytic and Schwannian differentiation, is
a pillar of diagnostic IHC. S-100 is also expressed by sus-
tentacular cells (eg, in pheochromocytoma/paraganglioma),
relevant to this discussion. The presence of S-100-positive
sustentacular cells has been touted as characteristic of an
appendiceal WDNET, especially in the differential diag-
nosis of an ileal tumor, which otherwise would stain sim-
ilarly for the markers already discussed. For example,
Lundqvist and Wilander234 described this pattern of S-100
expression of 11 of 12 (92%) appendiceal tumors and not in
small intestinal (0/11) or cecal (0/10) tumors. Similarly,
Moyana and Satkunam235 found sustentacular cells in 8 of
8 (100%) appendiceal WDNETs and 0 of 8 (0%) jejunoileal
ones. Sustentacular cells are also characteristic, although
less frequently seen, in bronchopulmonary carcinoids.
Barbareschi et al236 found them in 18 of 46 (39%) tumors;
As discussed above, most NENs, even poorly differ-
entiated examples, express SSTRs. Of these, the 2A subtype
is the most relevant, as the somatostatin analogues used
clinically (SRI, octreotide/lanreotide therapy, and peptide
receptor radionuclide therapy) have the greatest affinity for
this receptor. SSTR2A IHC may have a role in selecting
patients for somatostatin analogue therapy in instances in
which SRI is unavailable or is negative (particularly in
small tumors).45 Until recently, application was limited by
the fact that the best characterized antibody, which is
polyclonal, was not commercially available. A recently
developed, commercially available MoAb (clone UMB-1)
compares favorably to this antibody and gold standard
receptor autoradiography. Körner et al239 recently reported
optimization of a staining protocol with this antibody.
Heat-induced epitope retrieval in a pressure cooker with
citrate buffer was shown to perform best. Quality and

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Bellizzi
intensity of staining are evaluated, with only membranous
staining considered truly positive. Staining is either faint
(1 +), strong but with incomplete membrane staining (2 +),
or strong with complete membrane staining (3 +). The
group recommended that staining of >10% of tumor cells
(of any intensity) be considered positive; staining of 1% to
10% of tumors cells be considered inconclusive, with the
caveat that 1 + staining in this setting is likely negative;
and that no tumor staining be considered negative. The
>10% cutoff was 86% sensitive, 95% specific, and had a
95% positive predictive value for high receptor levels by
autoradiography. Of 27 tumors with no staining, 96% had
no or low receptor levels by autoradiography. In the future,
alternative somatostatin analogue–based therapies may be
developed that preferentially target other or a wider range
of SSTRs.
Vesicular Monoamine Transporter 2
Vesicular monoamine transporters (VMATs) are
integral membrane proteins involved in loading biogenic
amines into synaptic vesicles. Two human isoforms exist,
with VMAT2 expression in the tubal gut relatively
restricted to the histamine-producing, EC-like (ECL) cells
of gastric corpus mucosa.240 In contrast, only rare VMAT1-
expressing neuroendocrine cells are found in the stomach,
while the majority of neuroendocrine cells in the small and
large intestine are VMAT1 positive. As such, VMAT2 has
been advocated as a marker of ECL-cell gastric WDNETs.
VMAT2 IHC is considered a considerable improvement
over histamine IHC, which is practically limited by the low
histamine content of ECL cells and its rapid dissolution in
liquid fixatives.
Rindi et al241 reported diffuse, strong VMAT2
expression in 16 of 16 (100%) ECL-cell tumors, the pres-
ence of only rare VMAT2-positive cells in 12 of 21 (57%)
EC-cell (ie, serotonergic) tumors and 9 of 11 (82%) panc-
NETs, and absent expression (0%) in 5 gastrin, 4 soma-
tostatin, and 3 enteroglucagon-expressing tumors. Like
NESP55, VMAT2 expression is also characteristic of
pheochromocytoma [6 of 6 (100%) in this series; of note,
both VMAT 1 and 2 are highly expressed in the adrenal
medulla]. Similarly, Jakobsen and colleagues found
VMAT2 expression in 11 of 12 (92%) gastric WDNETs
and less frequent and generally focal expression in appen-
diceal (4/21; 19%), rectal (2/22; 9%), and pancreatic (8/24;
33%) tumors. VMAT2 expression was also detected in 34
of 52 (65%) ileal tumors, and in 7 (13%) it was diffuse.
Anlauf and colleagues performed a focused investigation of
non-neoplastic pancreas and pancNETs. In the non-neo-
plastic pancreas, VMAT2 expression was restricted to b
cells. It was also seen in 15 of 44 (34%) insulinomas and
12 of 48 (25%) other functioning and nonfunctioning
tumors.242 These studies serve to highlight high sensitivity
but lack of specificity for an ECL-cell primary (although
diffuse, strong expression may be moderately specific). In
fact, a recent study has suggested VMAT2 be regarded as a
“general neuroendocrine marker.”243 And unfortunately,
although high-level expression is typical of the ECL-cell
tumors driven by hypergastrinemic states (ie, those arising
in autoimmune atrophic gastritis and combined MEN1-
ZES), it is also usually encountered in sporadic gastric
WDNETs, as well.241,244,245 Given the evidence, VMAT2
expression appears to be more scientifically interesting than
diagnostically useful.
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Adv Anat Pathol  Volume 20, Number 5, September 2013
OTHER MARKERS OF INTEREST IN POORLY
DIFFERENTIATED NEUROENDOCRINE
CARCINOMAS
Merkel Cell Polyomavirus Large T Antigen
Drs Yuan Chang and Patrick Moore, having pre-
viously discovered the human herpesvirus that causes
Kaposi sarcoma, hypothesized that, given its epidemiology
(ie, associations with older age and immune suppression),
MCC might also be attributable to an infectious agent.
Utilizing a technique they dubbed “digital transcriptome
subtraction,” which entailed (1) sequencing tens of millions
of bases from cDNA libraries of 4 MCCs, (2) aligning those
sequences with National Center for Biotechnology Infor-
mation reference sequences, and (3) looking for homology
with infectious agents in the rare nonaligned sequences,
they discovered a single sequence with high homology to
known polyomaviruses.246 They subsequently identified the
virus, which they termed Merkel cell polyomavirus
(MCPyV) in 8 of 10 (80%) tumor samples. MCPyV is
clonally integrated into tumor DNA and is associated with
characteristic mutations in the large T antigen (LT Ag),
which abolish the virus’s ability to effectively replicate while
maintaining LT Ag’s oncogenic properties.247 To date,
MCPyV has been detected by polymerase chain reaction by
B20 groups analyzing B600 MCCs, generally in the 80%
range initially reported by Chang and Moore. A notable
outlier was a cohort of Australian tumors, in which virus
was detected in only 5 of 21 (24%).248
Chang and Moore’s group developed a MoAb to
residues 116 to 129 of the LT Ag (clone CM2B4), which has
emerged as a fairly sensitive and highly specific marker of
MCC. In 8 studies including 313 pure MCCs, LT Ag
expression has been detected in 60% (Table 10). As with the
polymerase chain reaction data, there is a single outlier,
again an Australian study in which only 19 of 89 (21%)
tumors were positive.253 Excluding this study, 168 of 224
(75%) tumors have been CM2B4 positive. About 5% of
MCCs are associated with actinic keratosis, squamous cell
carcinoma in situ, a component of squamous cell carcinoma
or basal cell carcinoma, or have foci of intratumoral squ-
amous differentiation (so-called “combined MCC”). Inter-
estingly, these tumors have been uniformly negative for LT
Ag (0/43; 0%). These findings suggest that, although most
MCCs are MCPyV related, a subset including the combined
tumors and some pure tumors are not, and are instead more
likely ultraviolet radiation–associated (explaining enrich-
ment in the Australian cohorts). Nearly 400 non-MCCs,
including a large number of PDNECs (predominantly
pulmonary but also CK20-positive tumors from the sali-
vary gland and uterine cervix) and hematolymphoid tumors
have been studied. Overall, 7 of 387 tumors (2%) have been
positive, including 6 of 23 (26%) SCLCs from 1 study.254
Outside of this 1 study, CM2B4 IHC has been extremely
specific, with only 1 other group reporting a single positive
case (of 74 SCNECs; 1.3%).253
The CM2B4 clone is now commercially available from
Santa Cruz Biotechnology (Santa Cruz, CA). It would
appear useful to support a diagnosis of MCC in the up to
10% of CK20-negative tumors and to argue against a
diagnosis of MCC in other CK20-positive SCNECs (espe-
cially primary parotid tumors). CM2B4 negativity appears
too frequent for it to exclude a diagnosis of MCC (espe-
cially in heavily sun-exposed populations). Rodig et al256
recently described a new MoAb (Ab3) raised against the
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TABLE 10. Merkel Cell Polyomavirus Large T Antigen Expression (CM2B4 Immunohistochemistry) in Merkel Cell Carcinoma and Other
Tumors
n (%)
References Country Pure MCC Combined MCC Other Tumors [Type]
Shuda et al249 Spain 21/36 (58) NE 0/146 (0) [142 hematolymphoid neoplasms,
4 PDNEC lung]
Busam et al250 USA 37/49 (76) 0/9 (0) 0/26 (0) [PDNEC lung]
Reisinger et al251 USA 15/20 (75) NE 0/12 (0) [nonmelanoma skin cancers in
MCC patients]
McCluggage et al177 Northern Ireland NE NE 0/4 (0) [PDNEC uterine cervix]
Chernock et al202 USA NE NE 0/7 (0) [PDNEC parotid]
Kuwamoto et al252 Japan 21/22 (95) 0/4 (0) NE
Paik et al253 Australia 19/89 (21) 0/15 (0) 1/74 (1.3)* [SCNEC, predominantly lung]
Jung et al254 South Korea 11/13 (85) NE 6/23 (26) [SCNEC lung]
Ly et al255 Canada 17/27 (63) 0/15 (0) 0/87 (0) [including 15 each of melanoma,
SCC, PDNEC lung, GI NET]
Rodig et al256 USA 46/57 (81) NE 0/8 (0) [SCNEC lung]
Range (median) (%) 21-95 (76) 0 (0) 0-26 (0)
Total 187/313 (60) 0/43 (0) 7/387 (2)
[all cases]
168/224 (75)
[non-Australian]
*The 1 positive non-Merkel cell tumor was a presumed lung primary.
GI NET indicates gastrointestinal neuroendocrine tumor; MCC, Merkel cell carcinoma; NE, not evaluated; PDNEC, poorly differentiated neuroendocrine
carcinoma; SCC, squamous cell carcinoma; SCNEC, small cell neuroendocrine carcinoma.

N-terminal 260 residues of the LT Ag with improved sen-
sitivity. Ab3 IHC was positive in 56 of 58 (97%) MCCs and
0 of 18 (0%) GI and lung PDNECs, while CM2B4 pos-
itivity was noted in only 81% of the same MCCs. Ab3’s
performance in an Australian group of tumors would be of
interest. The antibody is not yet commercially available.
Neurofilament
Neurofilaments (NFs) are 1 of 6 types of intermediate
filaments. NF IHC has applications in neuropathology and
soft tissue pathology, in which it is used to highlight axons.
NF IHC has been suggested as a marker of MCC, espe-
cially in its differential diagnosis with SCLC. In the 5
largest series directly comparing NF expression in MCC
and SCLC, expression has been noted in 67 of 93 (72%)
and 0 of 143 (0%) tumors, respectively.166,189,190,257,258
Expression is typically perinuclear/dot-like.
As a note of caution, NF expression has been detected by
some investigators in SCLC, and it can be seen in a subset of
extrapulmonary SCNECs. In 1983, Lehto et al259 first described
detection of NF expression in SCLCs (6/6, 100%; using a
rabbit polyclonal antibody) and actually suggested its utility in
distinguishing this tumor type from other lung tumors (all 22 of
which were negative). In a follow-up study, van Muijen et al260
failed to detect NF expression with 2 different MoAbs in a
series of 9 SCLCs. The largest study to detect NF expression in
SCLC was reported by Shy et al,261 who found 69% of 67
tumors to be positive (utilizing an MoAb from Immunotec).
These discrepant results may, at least in part, be attributable to
differences in the antibodies used to study NF expression.
Nagao et al157 detected NF in 6 of 15 (40%) SCNECs of
the major salivary glands, and McCluggage et al177 found NF
in 7 of 21 (33%) PDNECs of the uterine cervix. Interestingly,
at both of these sites primary PDNECs with a MCC-like
phenotype (ie, CK20 positive) are not uncommon, and while
Nagao and colleagues’ NF-positive cases coexpressed CK20,
McCluggage and colleagues’ did not. Otherwise, there are
little data on NF expression in extrapulmonary PDNECs.
Gaffey et al262 failed to detect NF in any (0%) of 19 color-
ectal tumors, as did Shin et al193 and Zamboni et al263 in 9
and 3 breast and ampullary tumors, respectively; most
recently, Lewis et al179 described expression in 1 of 14 (7%)
head and neck mucosal-based tumors.
Like MCPyV IHC, detection of NF may be useful
to support a diagnosis of MCC. Individual laboratories
would need to verify a lack of significant expression in SCLC.
Additional data on NF expression in extrapulmonary
PDNECs, some of which are clearly positive, are of interest.
APPROACH TO A SUSPECTED METASTATIC NEN
An algorithmic approach to a suspected metastatic
WDNET, with an emphasis on assigning site of origin, is
presented in Figure 9. The first considerations are to dis-
tinguish WDNET from other non-neuroendocrine tumor
types (eg, adenocarcinoma, squamous cell carcinoma) and
from PDNEC. As illustrated in Figure 1C, WDNETs, espe-
cially those with type C histology, are occasionally mistaken
for adenocarcinomas. I have a very low threshold for per-
forming IHC for broad-spectrum keratins (eg, AE1/AE3)
and general neuroendocrine markers (eg, synaptophysin and
chromogranin). As discussed above and illustrated in
Figure 3, WDNETs are sometimes mistaken for PDNECs,
especially in crushed small biopsies. Ki-67 IHC guards
against this pitfall and allows for assignment of WHO G1 or
G2 (with the caveats that this grading scheme only formally
applies to GEP tumors and that I have sometimes seen grade
discordance between metastases and matched primaries, with
the metastases generally of higher grade). For tumors with a
PI > 20% an alternative diagnosis (eg, PDNEC) should be
considered, although tumors with well-differentiated histol-
ogy but a PI in the G3 range have been described.131,264
There are very limited data on transcription factor IHC in
these rare tumors, although in 1 recent report Islet 1 was
expressed in 5 of 7 (71%) well-differentiated/G3 pancreatic
NENs versus only 1 of 13 (8%) pancreatic PDNECs.131

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Bellizzi
FIGURE 9. IHC workup of a metastatic WDNET of unknown origin. IHC indicates immunohistochemistry; NE, neuroendocrine; PI,
proliferation index; pPAX8, polyclonal PAX8; PR, progesterone receptor; PrAP, prostatic acid phosphatase; WDNET, well-differentiated
neuroendocrine tumor.
Once the diagnosis of metastatic WDNET is secured,
attention can turn to assigning site of origin. Any available
clinical information, especially the results of imaging studies, is
welcome. If the site of origin is “occult” at presentation, given
multiple lines of evidence, including the Duke biochemical and
survival data and the UCSF surgical exploration experience,
I believe a jejunoileal primary to be most likely. I examine the
tumor with this thought in mind, seeking out characteristic
histologic features including the presence of a monomorphous
population of round (rather than spindled) cells, perhaps
punctuated by an occasional larger nucleus; moderate (rather
than voluminous) amounts of cytoplasm, ideally with scattered
eosinophilic cytoplasmic granules; and disposition in a type A
pattern (although I have certainly seen types B, C, D, and
mixed patterns in metastases from midgut primaries).
For a metastatic WDNET of unknown origin, I per-
form CDX2, TTF-1, and polyclonal PAX8 IHC. For
tumors with a known or suspected origin, I may use 1 or
more markers to confirm the clinical impression (eg, only
CDX2 in a suspected midgut metastasis). I do not have Islet
1 IHC in my laboratory at this time, although in the
algorithm a positive reaction with the polyclonal PAX8
antibody or for Islet 1 are considered equivalent. I have
divided the results of this core panel into 4 patterns. The
presence of diffuse, strong CDX2 expression, with negative
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Adv Anat Pathol  Volume 20, Number 5, September 2013
results for TTF-1 and polyclonal PAX8 (or Islet 1) is strong
support for a midgut origin, while TTF-1 positivity, with
negative results for the other markers, strongly supports a
lung origin. Polyclonal PAX8 (or Islet 1) positivity is less
specific, as it may be seen with pancreaticoduodenal or
rectal tumors; it also may be accompanied by CDX2
expression, which is typically weak and patchy. Given
WDNET biology, though, I interpret polyclonal PAX8 (or
Islet 1)-positive tumors to be likely pancreatic in origin.
Upper and lower endoscopy can be pursued to exclude
duodenal and rectal primaries, respectively. The final,
“triple-negative” pattern (although CDX2 may actually be
“weak, patchy positive”) is noninformative. I report it as
“unusual for a midgut tumor (although it may be seen in up
to 10%) and in keeping with, although not ‘diagnostic of,’ a
pancreatic origin” (as up to half of the metastases from the
pancreas are polyclonal PAX8 negative—an argument for
bringing Islet 1 IHC online). For the final 2 patterns,
additional IHC is of interest, and I have occasionally
attempted PR or PrAP IHC in the setting of a non-
diagnostic immunophenotype. Finally, it is not unlikely
that additional markers will emerge in the near future that
will further inform the assignment of site of origin.
As with the well-differentiated tumors, the first step in
working up a PDNEC is to distinguish it from other tumor
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Adv Anat Pathol  Volume 20, Number 5, September 2013
types. This is especially important in the liver, where
metastatic adenocarcinomas are so frequent. Adenocarci-
nomas often contain minor populations of neuroendocrine
cells (on the order of 1% to 5% of total cells), and the
results of synaptophysin and chromogranin IHC are
sometimes “overinterpreted” in this setting. Occasional
poorly differentiated tumors will have extensive neuro-
endocrine marker expression (usually synaptophysin rather
than chromogranin), while lacking the histologic features of
a PDNEC. These have been referred to as “non–small cell
carcinomas with (occult) neuroendocrine differentiation.” It
is important to distinguish these from true PDNECs, as
they are unlikely to respond to “small cell” chemotherapy.
Although the histomorphology of SCNEC and LCNEC
differs, a common feature, and one that generally dis-
tinguishes them from other high-grade carcinomas, is
monomorphism.
In the skin, once the diagnosis of a PDNEC is made,
the key distinction is between MCC and metastatic visceral
PDNEC. This is usually easily accomplished with the mini-
IHC panel of CK20 and TTF-1. CK20 positivity essentially
confirms a diagnosis of MCC, while TTF-1 positivity
refutes it. Given negativity for both markers, correlation
with imaging studies is recommended, since, if a PDNEC is
metastatic to the skin, it is apt to be detectable elsewhere.
NF and MCPyV (CM2B4) IHC may also be helpful in this
setting. The latter of these may also be useful in the head
and neck, as parotid gland tumors are often CK20 positive
but MCPyV negative.
IHC is not useful in assigning the site of origin of a
visceral PDNEC. Pathologists often infer a lung origin
based on TTF-1 expression, but expression is also seen in
around 35% of extrapulmonary tumors. In fact, given a
morphologic impression of SCNEC and negative chromo-
granin and synaptophysin immunostains, I have occasionally
used TTF-1 expression to support the tumor diagnosis.
Regardless of the site of origin, metastatic PDNECs are
generally treated with “small cell” chemotherapy.
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