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Neuroendocrine Tumors of the Pancreas: Current Concepts

and Controversies

Michelle D. Reid &Serdar Balci &Burcu Saka &

N. Volkan Adsay

Published online: 16 January 2014# Springer Science+Business Media New York 2014

Abstract In the past decade, the clinico-pathologic charac-

teristics of neuroendocrine tumors (NETs) in the pancreas

have been further elucidated. Previously termed islet celltumors/carcinomasorendocrine neoplasms, they are now

called pancreatic NETs (PanNETs). They occur in relatively

younger patients and may arise anywhere in the pancreas.

Some are associated with von HippelLindau, MEN1, and

other syndromes. It is now widely recognized that, with the

exception of tumorlets (minute incipient neoplasms) that oc-

cur in some syndromes like MEN1, all PanNETs are malig-

nant, albeit low-grade, and although they have a protracted

clinical course and overall 10-year survival of 6070 %, even

low-stage and low-grade examples may recur and/or metasta-

size on long-term follow-up. Per recent consensus guidelines

adopted by both European and North American NET Socie-

ties (ENETS and NANETs) and WHO-2010, PanNETs are

now graded and staged separately, unlike previous classifica-

tion schemes that used a combination of grade, stage, and

adjunct prognosticators in an attempt to define benign be-

havior or malignant categories. For staging, the ENETs

proposal may be more applicable than CAP/AJCC, which is

based on the staging of exocrine tumors. Current grading of

PanNETs is based on mitotic activity and ki-67 index. Other

promising prognosticators such as necrosis, CK19, c-kit, and

others are still under investigation. It has also been recognized

that PanNETs have a rather wide morphologic repertoire

including oncocytic, pleomorphic, ductulo-insular, sclerosing,

and lipid-rich variants. Most PanNETs are diagnosed by fine

needle aspiration biopsy, in which single, monotonous

plasmacytoid cells with fair amounts of cytoplasm and dis-

tinctive neuroendocrine chromatin are diagnostic. Molecularalterations of PanNETs are also very different than that of

ductal or acinar tumors. Loss of expression of DAXX and

ATRX proteins has been recently identified in 45 %. Along

with these improvements, several controversies remain, in-

cluding grading, value of current cutoff ranges, and the best

methods for counting ki-67 index (manual count by computer-

captured image may be the most practical for the time being).

More important is the controversial use of the term carcino-

ma, which was previously employed in WHO-2004 only for

invasive and metastatic cases but has now been made synon-

ymous with grade 3 group of tumors. It is becoming clear that

grade 3 group comprises two distinct categories: (1) differen-

tiated but proliferatively more active tumors which typically

have ki-67 indices in the 2050 % range and (2) true poorly

differentiated NE carcinomas as defined in the lung, with ki-

67 typically >50 %. Further studies are needed to address

these controversial aspects of PanNETs.

Keywords Pancreas.Neuroendocrine tumor. WDNET.

PDNEC

Introduction

Many new developments have taken place in our understand-

ing of the biology, morphologic, and molecular characteristics

of neuroendocrine tumors of the pancreas. In 2010, the World

Health Organization (WHO) proposed a new terminology for

these tumors [1], which has had far-reaching effects on their

current diagnosis and management. As a very important and

positive development, the grading and staging parameters for

these tumors have now been separated from their name and

diagnostic sub-categories. In the ensuing article, we will

M. D. Reid :S. Balci : B. Saka :N. V. Adsay

Department of Pathology, Emory University School of Medicine,

Atlanta, GA, USA

N. V. Adsay (*)

Department of Pathology, Emory University Hospital, 1364 Clifton

Rd NE, Room H178, Atlanta, GA 30322, USA

e-mail: nadsay@emory.edu

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review these new developments, their limitations and contro-

versies, as well as recent advances in the diagnosis, classifi-

cation, and management of these tumors.

Definition and Terminology

Neuroendocrine tumors (NETs) of the pancreas are derivedfrom, and composed of, epithelial cells with phenotypic and

ultrastructural neuroendocrine differentiation. A spectrum of

neoplasms with neuroendocrine differentiation occurs in the

pancreas, ranging from well-differentiated to undifferentiated.

Differentiated examples are essentially counterparts of carci-

noid tumors elsewhere, now designated as well-differentiated

neuroendocrine tumors (WDNETs). The term carcinoid how-

ever has not been employed in the pancreas, except in the case

of metastases from the gastrointestinal tract or rare serotonin-

producing examples. Instead, in the past, pancreatic neuroen-

docrine tumors were termed islet cell tumors/carcinomasdue

to their resemblance to islets of Langerhans, the endocrinecomponent of this organ. However, it is now presumed that

these tumors do not necessarily arise fromthe islets, and in

fact, many may actually originate from the neuroendocrine-

type cells residing in the pancreatic ductal epithelium. They are

also regarded as a part of the age-old APUDoma concept

since they are fundamentally characterized by amineprecursor

uptake and decarboxylation activity. Such APUD cells have

endocrine, autocrine, paracrine, and neuromodulatory functions.

In the WHO-2004 classification system, these tumors were

referred to as pancreatic endocrine neoplasms. However, in

the WHO-2010 system, endocrine tumor was modified to

neuroendocrine tumorbased on the belief that the latter term

was a more accurate indicator of the cells neural as well as

endocrine phenotype and, more importantly, to distinguish

them from the conventional endocrine cells of the adrenal,

thyroid, and pituitary gland.

Although it is widely agreed that the term neuroendocrine

neoplasm would be a more accurate categorical designation for

theseneoplasms, the term NETwas adopted by the WHO in

2010 primarily because the acronym had already been incorpo-

rated into the very names of the societies dedicated to studying

them (i.e., the European Neuroendocrine Tumor Society

(ENETS) and the North American Neuroendocrine Tumor

Society (NANETs)). Thus, these tumors are currently referred

to as pancreatic neuroendocrine tumors (PanNETs). The acro-

nym P-NET is used by some authors; however, because the

latter can be confused with the primitive neuroectodermal tumor

acronym, PNET, the former designation is preferred.

Poorly differentiated neuroendocrine carcinomas are cur-

rently also included in the generic category of PanNETs and

are recognized as the high-grade (grade 3 [G3]) version of

these tumors. However many authors advocate making a

distinction between ordinary (well-differentiated) PanNETs

and these poorly differentiated neuroendocrine carcinomas,

regarding them as a separate category in the pancreas (see

the following discussion).

It should be noted that, unlike the WHO-2004 system, in

the WHO-2010 (Table1) as well as in current practice, the

terminology of NETs is no longer modified by the tumors

stage and grade. Previously, the term endocrine carcinoma

was employed for cases showing extra-pancreatic spread ormetastasis. However, this was problematic because a percent-

age of cases that were classified as benign demonstrated

malignant clinical behavior. This necessitated a shift in the

designation of cases that were originally classified as benign

but in whom metastases were discovered shortly after. While

this issue was resolved with the WHO-2010, another poten-

tially problematic terminology shift was inadvertently created

because in the latter classification carcinoma was made

synonymous onlywith G3 (poorly differentiated) NETs. Un-

fortunately some practitioners still use the term carcinoma

for metastatic NETs even when they are well differentiated

(G1 or 2 tumors).

Clinical Aspects

PanNETs are relatively rare tumors, accounting for 2 % of all

pancreatic neoplasms and affecting one to two individuals per

1,000,000/year. However, with improving technology, more

and more cases come to attention as incidentalomas [2].

Tumors are most frequently seen in adults but may rarely

occur in children. Patients are typically 3060 years (with a

mean of 50 years). Men and women are equally affected but

high-grade (G3) carcinomas often occur in older males.

PanNETs more commonly arise in the pancreatic head but

can involve any part of the pancreas.

WDNETs may also arise in a background of familial syn-

dromes including von HippelLindau (VHL) syndrome, tu-

berous sclerosis complex (TSC), neurofibromatosis type 1

(NF1), and multiple endocrine neoplasia type 1 (MEN1). In

Table 1 Classification system for pancreatic neuroendocrine tumors

Modified from WHO 2010

Differentiation Grade Mitosesa/10

|HPF

Ki-67b

proliferation

|index (%)

Well-differentiated PanNET Grade 1 20

WHOWorld Health Organization, HPFhigh power field, PanNETpan-

creatic neuroendocrine tumor,PanNECpancreatic neuroendocrine carci-

noma (large and small cell type)aIn at least 50 HPFsb MIB1 antibody

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MEN1 and VHL, tumors are frequently multifocal and occur

in a younger age group [3,4]. Up to 80 % of MEN1 patients

develop multiple ( 0.5 cm)

(Fig. 1) [3]. Up to 17 % of patients with VHL syndrome

develop PanNETs. Most of these are non-functioning and

60 % have clear cell morphology due to intracytoplasmic lipid[4,5]. Mutation of the VHLgene has been identified in these

patients and plays a role in disease development. Although

lipid-rich cells were once thought to be pathognomonic of

VHL-related WDNETs, similar cells have now been identified

in a subset of WDNETs that are either MEN1-related or non-

syndrome-associated [6]. Up to 10 % of patients with NF1

develop WDNETs, which have an increased affinity for the

ampullary region.

Cell Type and Functionality Status

Based on clinical presentation, PanNETs were historically

separated into two clinical categories, functional versus

non-functional, based on the symptoms elicited by the pre-

dominant hormone the tumors secrete into the bloodstream.

Clinical presentation and prognosis were once felt to be

strongly linked to tumor functionality. However, it has now

become clear that many tumors secrete multiple hormones

albeit with variable detectability and functional impact. Addi-

tionally, tumors may change their hormone productivity over

time. Moreover, with recent improvements in imaging modal-

ities, more non-functional PanNETs are being discovered, and

so the value of a functionality-based classification system has

lost support.

Functional tumors are still named based on primary clinical

presentation and serologic activity (e.g., gastrinoma and

insulinoma) and NOT on immunohistochemical hormone ex-

pression. Insulinomas often present with the classicalWhip-

ple triad due to excessive insulin production [7]. Gastrin

overproduction is associated with ZollingerEllison syn-

drome, while overproduction of vasoactive intestinal peptide

(VIP) can cause severe watery diarrhea, hypokalemia, andachlorhydria, leading to death.

Hormone status has also been shown to be associated with

biologic behavior, which is indirectly linked to the stage at

which a hormone-producing tumor causes clinical symptoms.

For example, most insulinomas have benign behavior, which

is largely attributable to their early detection. Over 70 % of

insulinomas are 2 cm, they are often

not detected early because glucagon levels are not clinically

elevated. Having said this, it is possible that cell type may

have additional yet to be defined biologic characteristics

that lead to divergent prognoses in these tumors.

Recently, small PanNETs occurring on the wall of pancre-

atic ducts, usually of serotonin-producing type, have come to

clinical attention due to duct obstruction sometimes mimick-

ing intraductal papillary mucinous neoplasms [8,9].

Classification and Sub-grouping

In the previous WHO-2004 classification system, PanNETs

had been sub-categorized as having benign behavior, as

having uncertain behavior, or as frank carcinoma (with

extrapancreatic spread or metastasis) based on an amalgam-

ation of grade, stage, spread status, and adjunct prognostica-

tors (vascular and perineural invasion). This was a fairly

unique approach for any type of tumor in the body. However,

in the WHO-2010 system, grade and stage were separated out,

as is the case for tumors of other organs. The latter is discussed

in detail in the following sections.

Grading

In the current 2010-WHO classification system, pancreatic neu-

roendocrine tumors are divided into well-differentiated [grade 1

(G1) and 2 (G2)] NETs (WDNETs) and poorly differentiated

(G3) neuroendocrine carcinoma (PDNECs). This system gives

the distinct impression that PDNECs are in continuum with

Fig. 1 In the background of atrophy and hyperplasticislets, there is an

evolving neuroendocrine tumor that is 3.0 mm in size (microadenoma)

(hematoxylin & eosin stain, 200 magnification). This patient had a

history of multiple endocrine neoplasia type 1 syndrome

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ordinary (well-differentiated) PanNETs, which is not neces-

sarily the case. Evolving evidence strongly suggests that G3

tumors should be regarded separately and as such will be

discussed separately later.

The grading of NETs is based on two calculations: (1)

mitotic count and (2) ki-67 labeling index (using the MIB1

antibody). G1 NETs are defined as having a ki-67 index of

50 %).

At the other end of the spectrum, for the distinction of G1

from G2 tumors, some groups advocate using 5 % as the cutoff,

which may be a more accurate and reproducible cutoff. While

further studies are needed to clarify this issue, the current 2 %

cutoff remains widely used. Additionally, the current cutoff

which is widely used and endorsed by the WHO-2010 is often

referred to as the 2 % cutoff in publications; however, as

recently clarified by NANETS, it is actually applied as

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prove to be a fairly reliable method. Of note, currently, there is

a separate billing code for automated counting. The direct

real-time eye-count approach, which is widely used in he-

matology, has also proven difficult in grading PanNETs, unless

a grid is used. However, even with a grid, cell distribution and

overlap make the test difficult to perform. We have found that,

in our current practice, the most practical, cost-effective, and

reproducible method for counting ki-67-positive cells is amanual count done either on a computer screen or as a cap-

tured static photomicrograph of tumor hot spots, where nega-

tive and positive tumor cells are visualized and immediately

crossed off once counted. The latter can be done with a digital

camera attachedto themicroscope, a setup that is far less costly

than automated methods and takes an average of 6 min [18].

Naturally, tumor heterogeneity and subjectivity in hot spot

determination are additional factors that may lead to variations

in results. Whether to count the less intensely labeled cells as

positive is also a question. For this, normalizationwith the

background tissue is helpful. There should be no background

staining in the stroma or cytoplasm in a properly performed ki-67 assay and light brown nuclei should generally be

disregarded. In tumors with abundant lymphocytes or other

confounding cell types, ki-67 indices may be falsely elevated.

In such cases, dual staining with neuroendocrine markers may

be necessary, especially if the labeling index appears to be

close to a cutoff, in particular 20 %.

Despite these challenges, ki-67 has nonetheless been

shown in numerous studies to correlate highly with clinical

outcome. Perhaps counter-intuitively, it has proven to be more

practical and reproducible than mitotic count, and is as, if not

more applicable, than other quantitative immunohistochemi-

cal assays that are the norm in other organs such as the breast

(ER/PR/her2neu) and stomach (EGFR).

Staging

The staging of PanNETs is now done separately from the

grade, unlike the WHO-2004 system where the two were

combined to determine the category.

There are currently several different staging protocols for

PanNETs. The one from The American Joint Commission on

Cancer (AJCC)/Union of International Cancer Control

(UICC)/College of American Pathologists (CAP) [1921]

differs from that of the European Neuroendocrine Tumor

Society (ENETS) [22] system (Table2). The AJCC has es-

sentially adopted the staging used for exocrine tumors, which

is problematic for pT3 tumors, which are defined in part by

peripancreatic soft tissue involvement. Because the pancreas

has very irregular lobules, no capsule, and fat placement

throughout, it is often difficult, if not impossible, to assess

peripancreatic soft tissueinvasion in these tumors [2325].

Furthermore, most PanNETs protrude from the pancreas even

when they are small, especially those located in the tail. That is

probably why many studies have failed to identify a correla-

tion between survival and T stage. In contrast, the ENETS

staging system relies predominantly on more reproducible

criteria such as tumor size rather than poorly defined and

irreproducible parameters like peripancreatic extension.

A recent study of 1,072 post-surgical PanNET patientsshowed that the ENETS system was superior to the

AJCC/UICC system for stratifying risk of death and creating

risk-based treatment guidelines [26,27]. Despite the proven

discrepancies between the two staging systems, there appears

to be no adverse effect on diagnosis and management [ 28]. In

our current practice, we typically provide the stage by both

schemes, separately.

Early PanNETsIncipient Neoplasia/Dysplasia

Extensive study of patients with MEN1 has led to the recogni-

tion of so-called precursor lesions of PanNETs. MEN1 patients

often develop proliferative or enlarged islets, some of which

acquire hormonal and morphologic clonality and achieve a size

beyond that of normal islets. The latter are regarded as dys-

plastic and are akin to pulmonary tumorlets. Naturally, it is

difficult to define the dysplasia and determine the point at

which such proliferations become autonomous and neoplastic.

These lesions often acquire an enveloping dense fibrous band,

a feature of neoplastic transformation commonly encountered

in endocrine organs. They also often show uniform staining

with pancreatic hormones. Lesions that appear fully established

but are

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It was in MEN1 syndrome that the idea of preneoplastic

precursors of pancreatic NETs was first fashioned as it gave

researchers the unique opportunity to study these tumors in

their earliest form. The term nesidioblastosiswas coined by

Laidlaw [30] who believed that pancreatic adenomas (islet cell

tumors) were a secondary reactive process in ductal epitheli-

um that occurred after an awakening of their islet and duct

bui lding capab ility. He nam ed these pluripotent cellsnesidioblasts (Greek for islet builder) and used the term

nesidioblastosis to describe islet cell proliferation and

nesidioblastomafor islet cell adenomas.

Others later proposed that nesidioblastosis might explain

the patterns of disease seen in familial multiple endocrine

adenomatosis (MEA) syndrome [31,32]. For almost a centu-

ry, nesidioblastosis has remained a part of the dysplasia/early

neoplastic transformation concept of PanNETs. Adult forms

of diffusenesidioblastosis (abnormality of cells) have also

been implicated in persistent hyperglycemia [33].

Characteristics of OrdinaryPanNETs (WDNET)

Fast-forward to the present day, where our knowledge of the

clinical aspects, biology, and associated alterations in these

tumors has expanded significantly: so how do we now view

and evaluate PanNETs?

Gross Features

PanNETs can involve any part of the pancreas. They are

typically well circumscribed and in sporadic cases are often

solitary. Their appearance may vary due to the contents of the

tumor. The vast majority are tan-brown and fleshy due to the

dense cellularity and relative stromal paucity (Fig. 2). Few

examples contain a more abundant stromal component and

may appear more yellow-white and firm/sclerotic. Some cases

have a complete capsule, but in most cases the capsule is

incomplete or non-existent. The vast majority of PanNETs

are solid tumors, but about 5 % present with cystic

degeneration, typically with clear serosanguinous contents,

and must be differentiated radiologically from cystic pancre-

atic tumors. Some examples may have extensive blood (so-

called peliotic variant) and appear grossly hemorrhagic.

PanNETs, especially those in the tail, often protrude from

the pancreatic surface and have a nodular bulge or polypoid

appearance on gross examination.

Histopathology

Unlike the more common pancreatic ductal adenocarcinoma,

PanNETs are mostly histologically stroma poor with morpho-

logic features that are dependent on their level of differentia-

tion. They are typically well circumscribed with variable

amounts of capsule-like tissue. In WDNETs, cells are ar-

ranged as sheets of monotonous epithelial cells, with anasto-

mosing cords, nests, and trabeculae, coarse salt and pepper

chromatin, and a fair amount of cytoplasm that may be eccen-

tric and plasmacytoid (Fig. 3). Glandular structures, tubulo-

acinar units, and pseudorosettes may also be seen, and thelatter may be so striking as to lead to misinterpretation as

solid-pseudopapillary neoplasms (SPNs). Mitotic activity is

usually scant. If mitoses are easily found, the possibility of an

acinar cell neoplasm or other high-grade tumor should be

excluded.

The correlation of morphologic findings with hormone

production (functionality status) is unreliable. Perhaps one

exception is the close association of psammoma bodies and

glandular pattern with immunohistochemical positivity for

somatostatin.

There are morphologic variants of PanNETs that are pecu-

liar and diagnostically challenging. The lipid-rich variant of

NETs is characterized by abundant foamy/microvesicular cy-

toplasm, which creates a picture virtually indistinguishable

from adrenocortical cells (Fig. 4). Some but not all of these

are associated with VHL syndrome. Interestingly, the cyto-

plasmic lipid frequently pushes and compresses the nucleus

peripherally, thus obscuring the classical nuclear features of a

NET, and may lead to their misdiagnosis as metastatic renal

cell carcinoma or adrenal tumor on morphology. Some

PanNETs have more uniform cytoplasm and may also show

prominent rhabdoid [34] or signet ring features. WDNETs are

also notorious for having oncocytic features characterized by

abundant granular, eosinophilic cytoplasm, and a single prom-

inent nucleolus, reminiscent of hepatocellular carcinoma

(Fig.5). When the oncocytic variant metastasizes to the liver,

it can be easily misdiagnosed as primary hepatocellular carci-

noma especially if appropriate immunohistochemical stains

are not performed. Unlike other morphologic variants, the

oncocytic PanNET has been found to be more aggressive in

some studies. Some tumors show a distinctsmall round cell

appearance and have a very high nucleus-to-cytoplasm ratio.

However, the nuclear molding, extensive necrosis, and high

Fig. 2 Well-differentiated pancreatic neuroendocrine tumor is seen aris-

ing in the pancreatic body as a solitary, well-circumscribed tan yellow

mass. Note the absence of hemorrhage and necrosis

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mitotic activity seen in the small-cell variant of PDNEC are

not typically seen. A peliotic variant of PanNETs is also

described, in which pools of blood and dilated blood vessels

are conspicuous both grossly and microscopically. Marked

cystic degeneration is seen in 5 % of tumors, and in such

cases, there is typically a cuff of ordinary PanNET on the cystwall, making the diagnosis rather straightforward.

PanNETs may show marked nuclear pleomorphism, so-

called endocrine atypia, similar to that seen in other endocrine

organs (Fig. 6). This can be so extensive that it can lead to

misdiagnosis as adenocarcinoma [35]. Tumors may rarely show

morphologic features mimicking those of paragangliomas [36].

Typically, these paraganglioma-like tumors show more

marked endocrine atypia (Fig. 7). The tumors presence in

the pancreas, its coexpression of keratin and neuroendocrine

markers, as well as the absence of basophilia and granularity

typically seen in paraganglioma help to clinch the diagnosis. In

some PanNETs with endocrine atypia, the degenerative

symplastic cells (akin to those seen in symplastic leiomyomas

or ancient schwannomas) can be so abundant and atypical that

the case can be misdiagnosed as a high-grade malignancy.

Some PanNETs contain abundant ductal elements, deemed

by some as the ductulo-insular variant of PanNET. The

ducts in such cases show obvious benign cytology. It is

debatable whether these are entrapped native ductules prolif-erating secondary to the local effects of the tumor or represent

an unusual transdifferentiation of tumor cells. The former

appears to be the more plausible explanation. These tumors

do not seem to behave differently than conventional PanNETs.

However, if the ductular elements have severe atypia, the

remote possibility of a mixed ductalneuroendocrine carcino-

ma ought to be considered, but these are exceedingly uncom-

mon tumors.

Although tumors are usually stroma poor, they may exhibit

marked sclerosis, especially those that secrete serotonin [37].

Necrosis is not a typical feature of PanNETs and, when

present, is usually focal, involves single cells, or may be

comedo-like. If there is extensive tumor necrosis, the

Fig. 3 a Well-differentiated

pancreatic neuroendocrine tumor,

grade 1. The tumor is stroma poor

with an organoid arrangement of

bland cells and intervening thin-

walled blood vessels

(hematoxylin & eosin stain, 400

magnification).b Tumor cells are

strongly positive for

chromogranin A (400magnification)

Fig. 4 Well-differentiated pancreatic neuroendocrine tumor, lipid-rich

variant. Tumor cells are large with abundant, clear, vacuolated cytoplasm

and oval nuclei lacking the salt and pepperchromatin typical of these

tumors (hematoxylin & eosin stain, 400 magnification)

Fig. 5 Well-differentiated pancreatic neuroendocrine tumor, oncocytic

variant. This example shows tumor cells with abundant granular eosino-

philic cytoplasm and nuclei with prominent central nucleoli (hematoxylin

& eosin stain, 200 magnification)

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possibility of an acinar cell neoplasm or high-grade neuroen-

docrine carcinoma ought to be considered.

Poorly Differentiated Neuroendocrine Carcinomas

(PDNEC)

In the WHO-2010, PDNECs are classified under the rubric of

grade 3 PanNETs along with proliferatively active ordinary

PanNETs. However, it is probably more accurate to regard

poorly differentiated neuroendocrine carcinomas (PDNEC) as

a separate entity or, at least, as the undifferentiated version of

differentiated PanNETs. These are essentially defined as theircounterparts in the lung. They are extremely aggressive tumors

that have often disseminated by the time of diagnosis. Median

survival is typically less than 2 years. Platinum-based therapeu-

tic agents have shown some promise in controlling their

growth; however, their overall prognosis remains grim.

These poorly differentiated, high-grade (G3) carcinomas are

subdivided based on cell size into small- and large-cell variants.

The large-cell variant is more common, often large, and charac-

terized by large cells with prominent nucleoli and variable

cytoplasm. Mitotic activity is brisk (often >40/10 HPFs) andnecrosis is often extensive. The small-cell variant shows small to

intermediate cells with coarsesalt and pepperchromatin, high

nucleus-to-cytoplasm ratio, inconspicuous nucleoli, prominent

nuclear molding, and crush artifact (Fig.8). Mitotic figures are

easily identifiable and there is extensive tumor necrosis.

Better delineation of PDNECs from ordinary G3 PanNETs

has been elucidated in some recent studies. Well-differentiated

but highly proliferative PanNETs typically have a ki-67 index

below 4050 % [10], whereas PDNECs typically show ki-67

>55 %, are characterized by highly aggressive behavior, and

require platinum-based therapy [11].

It should be noted here that PDNECs are very uncommontumors, and most cases that are diagnosed as such prove to be

acinar cell carcinomas once studied more carefully [38].

Ancillary Studies

Immunohistochemistry

PanNETs are essentially defined by the production of cyto-

plasmic neuroendo crin e granules, which are commonly

highlighted by immunohistochemical markers chromogranin,

synaptophysin, and CD56 (Fig.3). Ordinary well-differenti-

ated PanNETs tend to show stronger more diffuse staining

with neuroendocrine markers than PDNECs. Synaptophysin

is highly sensitive but less specific. Chromogranin is the most

specific neuroendocrine marker but is the least sensitive of the

three. CD56 antibody (123c3) which is directed against neural

Fig. 6 Well-differentiated pancreatic neuroendocrine tumor, pleomorphic

variant. Tumor cells show marked nuclear enlargement,pleomorphism, and

bizarre nuclei (hematoxylin & eosin stain, 400 magnification)

Fig. 7 Well-differentiated pancreatic neuroendocrine tumor with

paraganglioma-like features. Tumor cells show focal pleomorphism

and a prominent nested pattern reminiscent of a paraganglioma (hema-

toxylin & eosin stain, 100 magnification)

Fig. 8 Small-cell neuroendocrine carcinoma of the pancreas. Tumor

cells are arranged as tight groups and sheets of small cells with high

nuclear to cytoplasmic ratio (hematoxylin & eosin stain, 200

magnification)

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cell adhesion molecules is the least specific neuroendocrine

marker but is nonetheless useful in the differential diagnosis of

these tumors. Neuroendocrine markers are also positive in

large-cell carcinomas, but staining intensity and distribution

are not as robust as in WDNETs.

Additionally, NETs strongly express pancytokeratin as a

marker of epithelial differentiation. Cytokeratin 19 (CK19),

which is regarded as a marker of ductal lineage in the pancre-as, is sometimes expressed by PanNETs and has been found in

some studies to be a marker of more aggressive behavior

[1315]. Because of the latter, some authors advocate doing

a CK19 stain on all PanNETs. CD117 (c-kit) has also been

proposed as an independent adverse prognostic marker in

PanNETs; however, its use is not routine [16].

Focal acinar differentiation (with trypsin or chymotrypsin

positivity) has also been reported in PanNETs, but it is usually

present as isolated cells. Approximately 45 % of sporadic

PanNETs show loss of expression of DAXX and ATRX

immunostains, which correlates with mutations in the DAXX

andATRXgenes [39]. Loss of Dpc4, a common phenomenon inductal adenocarcinoma of pancreas, is only rarely (if ever) seen

in neuroendocrine tumors, which suggests that inactivation of

theSMAD4/DPC4gene is a rare event in these tumors.

CD99, a transmembrane glycoprotein encoded by theMIC-2

gene, and progesterone receptor (PR) are also positive in a

subset of PanNETs, in a manner similar to non-neoplastic islet

cells. The nuclear transcription factor islet-1 (isl1) is frequently

expressed in PanNETs [40, 41]. Pancreatic duodenal homeobox

1 (PDX-1), a homeodomain transcription factor, plays a regu-

latory role in early pancreatic development and is a marker of

pancreatic PanNET (in both primary and metastatic tumors).

Some studies have found isl1 and PDX-1 to be helpful in the

differential diagnosis of pancreatic NETs from non-pancreatic

WDNETs at metastatic sites such as liver, but their sensitivity

and specificity have not been rigorously tested [42].

Immunohistochemical analysis of PanNETs often lacks cor-

relation with serologic status and, in some cases, may yield

opposite results, presumably dueto therapid release and dispersal

of the hormone product without cellular accumulation, but with

retention of other hormones produced. Therefore, correlation

between functional status and immunohistochemical hormone

expression remains an imperfect science, and the use of immu-

nohistochemistry to assess hormone production is unreliable.

In a recent study, small- and large-cell PDNECs were shown

to be genetically related but distinct from ordinary PanNETs

[43]. P53 is positive in both small-cell (100 %) and large-cell

NECs (90 %) but is negative in well-differentiated PanNETs.

Rb protein is lost in 6090 % of PDNECs, and for tumors that

retain Rb, there is usually concurrent loss of p16 staining,

indicating that the two are mutually exclusive in NEC. Small-

and large-cell NECs show abnormal immunolabelling with p53

and Rb in 95 and 74 % of cases, respectively, which correspond

to intragenic mutatedTP53 and retinoblastoma RB-1 genes

[43]. Conversely, WDNETs retain Rb and p16. PDNECs retain

DAXX and ATRX immunostains, unlike ordinary PanNETs in

which these are frequently lost. PDX-1 is positive in up to 40 %

of NECs, with large-cell tumors showing more positivity.

PAX8 is positive in 50 % of NETs (including PDNECs and

WDNETs).

BCL2 protein, which is overexpressed in small-cell carci-

noma of the lung, is also overexpressed in PDNECs (100 % ofsmall-cell and 50 % of large-cell tumors) but is negative in G1

tumors and variably expressed in G2 WDNETs [43]. The

platinum-based chemotherapeutic agents used to treat small-

cell lung carcinoma exert their effects by inducing apoptosis

via a BCL2 regulated pathway. This suggests that BCL2

expression by PDNECs could potentially be exploited with

BCL2 antagonists similar to those used in the lung.

Electron Microscopy

On ultrastructural analysis, NETs contain membrane-bound,

dense core neurosecretory granules, which historically havebeen highly useful in their diagnosis. However, ultrastructural

analysis is no longer accessible in most institutions and has

been superseded by immunohistochemistry. Electron micros-

copy may be useful in very poorly differentiated tumors or

cases with an amphicrine phenotype.

Molecular Studies

PanNETs associated with MEN1 and VHL syndromes show

mutations in theMEN1 andVHL genes. In MEN1, theMEN1

(menin) gene located on chromosome 11q13 is mutated.

Among sporadic PanNETs, somatic MEN1 mutation or loss

of heterozygosity at the MEN1locus is seen in a significant

number of cases [39, 4447]. MEN1 mutation is associated

with a better prognosis. PDNECs however are infrequently

associated with MEN1[1]. Deletion of theVHLgene occurs in

up to 25 % of sporadic WDNETs.

Among sporadic PanNETs, inactivating somatic mutations

of the DAXX(death-domain associated protein) and ATRX

(alpha thalassemia/mental retardation syndrome X-linked)

genes have been identified in 45 % of cases. DAXX and

ATRX proteins are crucial for telomere maintenance [48],

and their associated genes are implicated in chromatin remod-

eling [39]. When mutated, DAXXand ATRXresult in immu-

nohistochemical loss of their corresponding proteins.

Approximately 15 % of PanNETs show genetic mutations

in the mammalian target of rapamycin(mTOR) cell signal-

ing pathway proteins [39]. The mTOR pathway alteration

represents a unique therapeutic target for drugs like everoli-

mus which are directed specifically at this pathway [49].

Chromosomal gains and losses have also been identified in

PanNETs, and multiple chromosomal abnormalities are asso-

ciated with a worse prognosis [50].

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Differential Diagnosis

Differential diagnoses of PanNETs include all primary pan-

creatic neoplasms with diffuse, cellular sheet-like proliferation

as well as metastatic tumors.

Acinar cell carcinoma is one such primary pancreatic tumor

that is derived from exocrine acinar cells and shows acinar

differentiation as evidenced by positivity for trypsin, chymo-trypsin, lipase, other pancreatic enzymes, and BCL10. Tumor

cells have abundant, granular, zymogen-rich cytoplasmic gran-

ules, coarse chromatin, and prominent, sometimes cherry red,

central nucleoli. A subset of acinar cell carcinomas is morpho-

logically almost indistinguishable from PanNETs. In addition,

most acinar cell carcinomas focally express neuroendocrine

markers either as scattered individual cells or large zones or

even as a very-well-formed distinct component (mixed carci-

nomas). The presence of exuberant mitotic activity in the setting

of seemingly bland monotonous epithelial cells is more in

keeping with acinar cell carcinoma than a differentiated

PanNET, in which mitotic figures are often few and far be-tween. Additionally, acinar cell carcinomas show prominent

central nucleoli, which are typically less prominent in

PanNETs. Most helpful though is the neuroendocrine chroma-

tin pattern. Morphologic distinction is facilitated by immuno-

histochemistry. Acinar cell carcinoma is negative or only focal-

ly positive for neuroendocrine markers and shows positivity for

pancreatic enzymes, while NETs show opposite results.

Pancreatoblastomas are morphologically similar to acinar cell

carcinoma and may also be confused with PanNETs. However,

immunohistochemistry as well as the finding of classical

squamoid morules should facilitate distinction.

Morphologic distinction of mixed acinarneuroendocrine

carcinoma, an acinar cell neoplasm that shows over 30 %

neuroendocrine differentiation, can be especially challenging

both on morphology as well as immunohistochemistry as

tumor cells stain with both acinar and neuroendocrine

markers. The key to distinguishing this variant from PanNET

is the mixed neuroendocrine and acinar immunophenotype as

well as the distribution of the neuroendocrine component,

which should not be significant in these mixed tumors.

Another key differencel is SPN. Both PanNET and SPN can

show solid sheet-like pattern and nested growth (Fig.9). Typ-

ically, the nesting is more vague in SPNs, and both patterns

blend, rather imperceptibly, with each other, creating a more

mesenchymal-like appearance. Striking overlapping of tumor

nuclei is more prominent in SPN and the presence of large

cytoplasmic vacuoles also favors SPN. On close examination,

one should note the absence of the salt and pepperchromatin

of neuroendocrine tumors and the presence of fine, open,

powdery chromatin along with longitudinal nuclear grooves,

which are distinctive findings in SPN. In addition, the presence

of PAS-positive, diastase-resistant hyaline globules is support-

ive of SPN, although similar globules can occasionally be seen

in PanNETs [51]. Immunohistochemistry is extremely helpful

in making the diagnosis as SPNs, in contrast to PanNETs, show

only focal or weak positivity for keratin and chromogranin and

strongly express nuclear-catenin. It should be kept in mind

that SPNs may express neuroendocrine markers CD56 and

synaptophysin, sometimes diffusely. This is a potential source

of misinterpretation of immunohistochemistry unless one pays

close attention to the distinguishing morphologic features. Itshould also be noted that some PanNETs exhibit striking

pseudopapillary pattern mimicking SPNs.

The clear cell or lipid-rich variant of WDNET can poten-

tially be misdiagnosed as metastatic clear-cell renal cell carci-

noma (RCC) involving the pancreas. Metastasis of clear-cell

RCC to the pancreas is a known phenomenon and is one of the

most frequent tumors to metastasize to the pancreas [52].

Additionally, the lipid-rich variant of WDNET often fails to

show the classical nuclear features of neuroendocrine neo-

plasms, making diagnosis more challenging (Fig.4). As men-

tioned earlier, the oncocytic variant of WDNET may resemble

hepatocellular carcinoma (HCC) and, if it first presents as livermetastasis, can be misdiagnosed as HCC.

Plasmacytomas are known to involve the pancreas, and for

WDNETs showing marked plasmacytoid features, distinction

from these hematopoietic neoplasms can be especially challeng-

ing. It is important to note that in plasmacytoma, the chromatin

distribution has a clock-facepattern with a distinctive perinuclear

Hof. In WDNET, the chromatin distribution is different and the

perinuclear Hof is absent. Plasmacytomas stain positively for

CD138 and show clonal immunoglobulin, light chain restriction,

with negativity for keratin and neuroendocrine markers.

The large-cell variant of poorly differentiated (G3) neuro-

endocrine carcinoma resembles poorly differentiated adeno-

carcinoma and may be misdiagnosed as such unless suspected

on morphologic assessment. Immunohistochemical expres-

sion of neuroendocrine markers in addition to keratin supports

the former. The small-cell variant of PDNEC may resemble

metastatic small-cell carcinoma of lung. TTF1 stain is not

helpful in distinction as small-cell carcinoma is TTF1-

positive in a variety of extra-pulmonary sites. Clinical infor-

mation and a history of previous carcinoma are especially

important in the accurate diagnosis of such cases.

The crushed and molded tumor cells of small-cell PDNEC

may resemble a high-grade lymphoma. Morphologic distinc-

tion is often impossible and immunohistochemistry is required

for diagnosis.

Small, round, blue cell tumors may involve the pancreas

primarily or secondarily. These include desmoplastic small,

round-cell tumor (DRCT) [53 ] as well as primitive

neuroectodermal tumors (PNETs) [54]. The latter also ex-

presses keratin in addition to CD99, both of which are also

expressed by WDNETs. However, the salt and pepperchro-

matin distribution is absent in DRCT and PNETs, which also

show characteristic molecular alterations (EWS-WT1 gene

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fusion and t(11;22), respectively) that help in distinction.

Merkel cell carcinoma may rarely metastasize to the pancreas

and in one report was present with a synchronous PanNET(insulinoma) [55]. Awareness of a previous history of this

disease should prompt appropriate immunohistochemical

studies to exclude this differential.

Because of their brisk mitotic activity and morphologic

resemblance to neuroendocrine cells, acinar cell carcinoma

may be mistaken for large-cell neuroendocrine carcinoma. A

panel of immunohistochemical stains including both neuro-

endocrine and acinar markers (trypsin, chymotrypsin) can

help to distinguish the two.

Cytology

Fine needle aspiration (FNA) has been used in the cytologic

diagnosis of primary and metastatic PanNETs for many years. It

is typically performed by endoscopic ultrasound-guided FNA,

which is successful at identifying NETs in up to 90 % of cases.

The cytologic features of these tumors are distinctive. For

ordinary, well-differentiated (G1 and 2) NETs, tumor cells are

classically singly dispersed or clustered as bland-appearing

monotonous cells with round to oval nuclei and variable cyto-

plasm, which may or may not have distinctive plasmacytoid

features (Fig.10). In addition, multiple cytoplasmic vacuoles

may be seen. On Papanicolaou stain, tumor cells show the

classical salt-and-pepper chromatin distribution, which sup-

ports the diagnosis (Fig. 10). Pseudorosettes are visible not only

on cellblock but also on smears. Immunohistochemical analysis

of cellblock material often demonstrates tumor cell positivity

for keratin as well as neuroendocrine markers.

In poorly differentiated neuroendocrine carcinoma of small-

cell type, the cytologic features are similar to the lung counter-

part. Tumor cells are often crushed and small to intermediate in

size with irregular nuclear borders, high nucleus-to-cytoplasm

ratio, crushed artifact, and nuclear molding. There is extensive

single-cell and background necrosis in addition to brisk mitotic

activity seen both on smears and cellblock. Tumor cells expresspancytokeratin as well as neuroendocrine markers, which help

to support the diagnosis. For large-cell neuroendocrine carcino-

ma, tumor cells are usually large with variable cytoplasm as

well as prominent central nucleoli. On cytology, these tumors

may resemble a poorly differentiated adenocarcinoma and may

not show the classical salt-and-pepper chromatin of WDNETs

or the nuclear molding of their small-cell counterpart. If a

neuroendocrine primary is not suspected at the time of evalua-

tion, this diagnosis may be easily missed.

The grading of PanNETs has been performed on FNA by

immunolabelling cell blocks with ki-67 [56, 57]. One might

argue that cytologic samples are notoriously hypocellular and

therefore lack the requisite minimum of 500 tumor cells

Fig. 9 Solid pseudo-papillary neoplasm of the pancreas mimicking a

pancreatic neuroendocrine tumor.a There is an organoid arrangement of

(stroma-poor) tumor cells with morphology similar to a well-differentiat-

ed pancreatic neuroendocrine tumor (hematoxylin & eosin stain, 100

magnification). b Tumor cells shownuclear and cytoplasmic positivityfor

-catenin immunostain (shown on the right) in contrast to the acinar cells

on the left, which do not express nuclear-catenin (200 magnification)

Fig. 10 Fine needle aspiration of a well-differentiated pancreatic neuro-

endocrine tumor showing singly dispersed, bland tumor cells with

plasmacytoid morphology, oval nuclei, and salt and pepperchromatin

(Papanicolaou stain, 400 magnification)

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required for calculation. Additionally, since NETs are fraught

with proliferative heterogeneity, is the index/grade generated by

an FNA (or even a core biopsy) reliable? Few have adequately

addressed this question, but there are studies that support grad-

ing of these tumors on biopsy-generated samples despite

intratumoral heterogeneity [58]. In a recent cytologic study,

grading results obtained by manual counting of ki-67-positive

cells correlated positively with indices obtained by automatedimage cytometer, as well as corresponding resections [56].

These findings suggest that the grading of PanNETs on cyto-

logic material is applicable, and in situations in which such

samples are the only ones available for evaluation (particularly

in patients with metastatic disease), they may present the only

opportunity for classification and treatment stratification.

Biologic Behavior

Ordinary PanNETs (G1 and G2) are considered low-grade

malignancies (with the exception of tumorlets described ear-lier), with an overall 10-year survival of 6070 %. In the

previous (WHO-2004) classification system as well as earlier

studies, an attempt was made to recognize a benign behav-

iorcategory. However, after more recent studies with longer

clinical follow-up, it became clear that even the incidentally

detected and resectable PanNETs that are 95 %.

The prognosis of the G3 category is more complicated,

largely due to the fact that this category encompasses at least

two distinct entities: ordinary PanNETs with high proliferative

rate and true PDNECs (Fig. 11). Preliminary results from

Basturk et al. indicate that G3 cases with ordinary PanNET

morphology are clearly more aggressive (median survival 32

months, 5-year survival 22 %) than G2 NETs (median survival

63 months, 5-year survival 61 %) but less aggressive than

PDNEC (median survival 15 months, 5-year survival 17 %)

[10]. Similar differences in survival have been shown by

others [10, 11, 59, 60]. This was also confirmed by a study

from Europe which showed that for all gastrointestinal G3

NECs survival times were longer when the ki-67 index was

2055 versus over 55 %, and the latter group was also moreresponsive to platinum-based therapy [11]. Accordingly, we

currently regard G3 NETs as two distinct groups: (1) well

moderately differentiated PanNET with proliferative index of

G3, which typically has ki-67 >20 and 50 %.

Treatment Options

It is generally agreed among experts that, if possible, all

functioning PanNETs should be resected [61, 62], with theexception of patients with MEN1 and ZollingerEllison syn-

drome or those with small (

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resection (cryosurgery) and transplantation, and hepatic artery

embolization or radiofrequency ablation for non-surgical

candidates.

The role of chemotherapy (or targeted therapy) has been

debatable. Since PanNETs are slow-growing tumors, they

often do not respond to conventional cytotoxic chemothera-

peutic agents. Recently, targeted therapies have become a

serious alternative consideration. For tumors with alterationsof the mTOR pathway, the drug everolimus has shown prom-

ise as a therapeutic agent [63] and is currently in phase IV

clinical trials (http://www.nanets.net/research/current-clinical-

trials). Cabozantinib, a tyrosine kinase inhibitor, and sunitinib,

a vascular endothelial growth factor inhibitor, have also

shown similar promise (http://www.nanets.net/research/

current-clinical-trials).

For patients with PDNECs, cisplatin and etoposide-based

regimens are recommended and patients often show 4070 %

(albeit brief) response rate [61]. The recent demonstration of

BCL2 overexpression by PDNECs [43] suggests that BCL2

antagonists may prove useful in their treatment in a mannersimilar to their current use in small cell carcinoma of lung,

which also expresses BCL2.

Summary of Key Elements to Include in a Pathology

Report

Resection of a Primary Pancreatic Neuroendocrine Tumor

Diagnosis and differentiation: well-differentiated PanNET

versus poorly differentiated PanNEC

Tumor grade: based on mitoses/10 HPF and ki-67 prolif-

eration index (%)

Tumor size

Lymphvascular invasion

Perineural invasion

Large vessel invasion

Margin status (for resections)

TNM stage (if a resection); specify method used (CAP/

AJCC vs ENETS)

Comment: Tumor grade, mitotic count/10 HPF, ki-67 index

(%), necrosis, neuroendocrine marker expression, cytokeratin

19 expression (optional)

Resection or Biopsy of Metastatic Tumor (Presumed)

from Pancreas

Diagnosis and differentiation: well-differentiated PanNET

versus poorly differentiated PanNEC

Margin status: for resections

Comment: tumor grade [based on mitoses/10 HPF, ki-

67 proliferation index (%)], additional (optional) stains

(PDX-1, isl1)

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