Introduction
Why this document
Neuroendocrine neoplasms (NENs) can arise almost throughout the entire body and share
common morphological, ultrastructural, and immunohistochemical characteristics.
Neuroendocrine neoplasms are an emerging entity that can occur at any age, with the
median age at diagnosis in the late fifth decade and an age-related incidence increase.
About two-thirds involve the gastro-entero-pancreatic (GEP) tract and epidemiological
studies show their increasing incidence [1]. In the last decades, the overall reported
incidence of GEP-NENs increased from 1.0 to 5.25/100.000 persons/year, with a present
estimated prevalence of 35/100.000 [1–10]. Physicians’ awareness, endoscopic screening
and increased sensitivity of diagnostic tools may at least in part explain this growing
trend.
Most guidelines are focused on staging, treatment and follow-up of NENs. However,
an appropriate clinical suspicion and a correct diagnostic work-up are critical starting
points. A multidisciplinary approach, moreover, is crucial to provide a timely and
integrated care. Hence, this document is neither a review, nor a guideline; rather,
it is a clinical guide for a stepwise and integrated diagnostic work-up of GEP-NENs.
Hopefully, this will result in a correct utilization of resources and optimization
of the cost/benefit ratio.
Methodology
The grading of recommendations, assessment, development, and evaluation (GRADE) system
was adopted for the present position statement [11–14]. Briefly, the GRADE system
classifies evidence into four quality levels (high, moderate, low, or very low), and
recommendations into two grades (strong or weak).
Whenever possible, the level of evidence (LoE) has been ranked as follows: very low
(⊗○○○), low (⊗⊗○○), moderate (⊗⊗⊗○), and high (⊗⊗⊗⊗). “Very low quality” evidence
corresponds to unsystematic clinical observations (case report, case series) or indirect
evidence (e.g., surrogate end points); “low quality” evidence corresponds to observational
studies or randomized controlled trials (RCT) with major limits; “moderate quality
evidence” corresponds to RCTs with limitations or rigorous observational studies;
and “high quality evidence” corresponds to well performed RCTs and strong evidence
from unbiased observational studies [13].
We labeled as “recommendations” and “suggestions” the strong and weak recommendations,
respectively. Each recommendation/suggestion is based on the quality of supporting
evidence, downgraded or upgraded according to adjunctive factors (e.g., inconsistency
of results, indirectness of evidence, lack of precision and limited number of relevant
publications downgrade the recommendation/suggestion; large effect size, narrow confidence
intervals, clinically very significant end points upgrade the recommendation/suggestion),
and the level of panel agreement [13].
Definitions
Neuroendocrine neoplasms neoplastic cells possess features of both neural and epithelial
cells. Therefore, in line with the WHO classification, the term neuroendocrine will
be adopted throughout this document [15].
WHO recommends the use of the term “neuroendocrine neoplasm” (NEN) to indicate low-
to high-grade lesions. The term “neuroendocrine tumor” (NET) will be used throughout
this document, due to its widespread diffusion, to indicate low- to intermediate-grade
lesions and the term “neuroendocrine carcinoma” (NEC) to indicate high-grade lesions.
Terms like “carcinoids” and the embryological classification of GEP-NENs in tumors
of foregut (thymus, esophagus, lung, stomach, duodenum, pancreas), midgut (appendix,
ileum, cecum, ascending colon) and hindgut (distal colon and rectum) will be avoided.
Classification
In the last 10 years WHO has repeatedly revised the pathologic classification of GEP-NENs
(Table 1) [16].
Table 1
WHO classifications of GEP-NENs
WHO 1980
WHO 2000
WHO 2010
I. Carcinoid
Well-differentiated endocrine tumorWell-differentiated endocrine carcinomaPoorly differentiated
endocrine carcinoma/small-cell carcinoma
Neuroendocrine tumors NET G1 (Grade 1) NET G2 (Grade 2)Neuroendocrine carcinoma NEC
G3 (Grade 3): Large-cell NEC small-cell NEC
II. MucocarcinoidIII. Mixed carcinoid-adenocarcinoma forms
Mixed exocrine–endocrine carcinoma
Mixed adeno-neuroendocrine carcinoma (MANEC)
IV. Pseudotumor lesions
Tumor-like lesions
Hyperplastic and preneoplastic lesions
According to the 2010 classification, NET G1 includes the “carcinoids” or “well-differentiated
tumors” of the 1980 and 2000 WHO classifications. These tumors are usually indolent,
but can occasionally behave as malignant.
NET G2 may be considered a “grey zone”, with heterogeneous behavior, and requires
a tailored management.
NEC (G3) is a malignant neoplasm with an aggressive clinical course.
MANEC has a malignant phenotype with features of both adenocarcinoma and NET. This
definition requires the presence of at least 25 % of each component. Neuroendocrine
cells are usually interspersed and the two populations may be identified only by immunohistochemistry
(IHC). Less frequently, neuroendocrine cells may be grouped in distinct regions that
are recognized by light microscopy.
The WHO 2010 classification strongly relies on tumor grading. Grading relates to the
biological aggressiveness of the neoplasm, whereas differentiation indicates its similarity
to the tissue of origin [15]. The clinical behavior of NENs may be basically predicted
by their grading, staging, and evidence of hormonal syndromes. All these data should
be collected and weighted to establish the prognosis and management of the patient.
Grading assessment
The grade of a tumor is the primary predictor of its clinical outcome. Grading is
based on the proliferation rate of the tumor, as assessed by the Ki-67 cell labeling
and by the mitotic count (number of mitosis × 10 high power fields—HPF) (Table 2)
[15–23].
Table 2
Grading system for GEP-NENs (adapted from 19)
Ki-67 index (%)a
Mitotic count/10 HPFb
NET G1
≤2
<2
NET G2
3–20
2–20
NEC G3
>20
>20
aAssessed by MIB-1 labeling in at least 2,000 tumor cells in high nuclear density
(“hot spot”) areas
b10 HPF = 2 mm2, at least 50 optical fields in high-density mitotic areas
Visual estimates are currently used as the standard technique for evaluating both
Ki-67 and the mitotic count [24, 25]. Several areas should be assessed within the
tumor to reduce the risk of evaluation bias due to intratumoral heterogeneity. Densely
stained regions (“hot spots”) should be preferentially evaluated. Results from these
areas should be reported as a single percentage reflecting the highest identified
count [16, 21, 22].
Potential pitfalls and limitations are:
technical problems (e.g., tissue processing, differences in Ki-67 antibodies, etc.);
intratumoral heterogeneity and sampling limitations (e.g., a single biopsy sample
may not be representative of the tumor grade within the whole neoplastic mass) [24,
26];
discordances:
I.
between the proliferative rate and the degree of differentiation (e.g., a morphologically
well-differentiated NEN may exhibit a high proliferative rate);
II.
between the predictive value for prognosis and that for treatment response (e.g.,
Ki-67 is a reliable predictor of disease progression and overall survival (OS), but
seems a less efficient predictor of response to medical treatment) [27].
Pathologic staging
GEP-NENs are staged according to tumor size, site of origin, and locoregional or distant
spreading [21–23]. The staging information is integrated with the 2010 WHO classification
to stratify the prognostic risk and optimize the therapeutic and follow-up strategies
(Fig. 1).
Fig. 1
Integrated pathologic and biologic classification (modified from 15)
Diagnostic tools
Histology, cytology, immunohistochemistry, and molecular biology
Morphologic criteria
Pathologic assessment is required for the diagnosis, classification and staging of
NENs.
GEP-NENs present a broad architectural spectrum [28]. Well-differentiated tumors show
an organoid pattern that ranges from solid nests to micro–macrotrabecular/gyriform
pattern. A rich sinusoidal vascularity is usually observed. Stromal fibrosis, amyloid
deposition, and calcification may be present. Necrosis can be present either as large
infarct-like areas or as punctate foci in the center of neoplastic nests. Regardless
of their growth pattern, NEN cells have a similar cytological appearance: small- to
medium-size cells with round to oval shape and eosinophilic, lightly granular, cytoplasm.
The nuclei are usually centrally placed, fairly uniform, with a finely dispersed (“salt
and pepper”) chromatin pattern. Rarely, the neoplastic cells have a “plasmocytoid
appearance” due to peripherally located nuclei. Nucleoli are usually inconspicuous
or absent. Intracytoplasmatic hyaline globules or nuclear pseudoinclusions may be
seen.
High-grade NENs are composed of small or large-to-intermediate cells with high-grade
features (marked nuclear atypia, multifocal necrosis, high mitotic index) and diffuse
growth, sometimes with organoid feature resembling NEN.
The subgroup of GEP-NENs with Ki67 >20 % (and therefore G3 according to WHO 2010),
but with a morphology of well-/moderately differentiated tumor should be considered
low/intermediate rather than high-grade NENs [29].
Cytological specimens, which may be the only source of diagnostic material, pose some
problems for clinical management. Cytology effectively separates high-grade NENs from
low-grade NENs, but the distinction between low- and intermediate-grade NENs may be
impossible. The diagnostic accuracy of aspiration techniques may be limited by the
small sample size, the suboptimal reproducibility and the risk of contamination from
contiguous tissues.
Cytology is gaining a major role for the diagnosis in duodeno-pancreatic tumors. The
endoscopic ultrasonography (EUS) fine needle aspiration (FNA) technique appears reliable,
with a reported specificity of about 75 %, sensitivity of 87.5 %, accuracy of 89 %,
positive predictive value (PPV) of 93 %, and negative predictive value (NPV) of 60 %
[30–32].
Immunohistochemistry and molecular biology techniques
Neuroendocrine differentiation Synaptophysin (a small vesicle-associated marker) and
Chromogranin A (CgA, a large secretory granule-associated marker) are useful IHC markers
for the diagnosis of NENs. In NEC, the staining for both these markers is required
to confirm the diagnosis, because CgA may be negative [15]. Routine IHC staining for
peptide hormones and bioamines is not recommended. Other neuroendocrine markers, such
as PGP.9.5, NSE, CD56, NSP-55, are of questionable specificity and clinical usefulness.
Prognostic markers proposed in addition to Ki-67 are CK19, CD117, CD99, p53, Her/2,
CEACAM1, E-cadherin, β-catenin, hHAS-1, FGF13, PLGF, PAX-8, PTEN. None is presently
recommended for clinical practice. The research of circulating tumor cells or the
use of microRNAs is not indicated for routine use [33, 34].
Markers of primary site These markers may be a key for determining the unknown primary
tumor in metastatic lesions. The most useful are [35]:
TTF-1, indicative of pulmonary or thyroid origin;
serotonin and CDX-2, indicative of intestinal origin;
PAX-8 and histidine-decarboxylase, indicative of pancreatic origin;
xenin, indicative of duodenal origin.
Markers predictive of response to specific treatments These biomarkers are not indicated
for routine diagnostic practice. They include [19]:
somatostatin receptors (SSTR)-2A (IHC determination at the cell membrane level), for
planning the treatment with somatostatin analogs (SA);
Akt/mTOR pathway molecules (PIK3, PTEN, TSC2), for treatment with everolimus;
thymidylate synthase, for treatment with antifolates;
ERCC-1, for treatment with platinum;
topoisomerase Iiα, for treatment with etoposide;
epigenetic events, as methylation of MGMT promoter, for treatment with alkylating
agents.
Working with the pathologist and his pathologic report
The modality and timing of sampling techniques should be planned by a multidisciplinary
team.
The pathologist should be provided with accurate clinical information including signs
and symptoms, laboratory findings and imaging studies [36].
The ideal pathologic report should include:
description of the macroscopic specimen;
tumor size (three dimensions);
description of cell features and histologic architecture;
differentiation (well or poorly differentiated);
IHC findings (CgA and synaptophysin routinely, SSTR2A when appropriate (e.g., when
functional imaging for SSTR2 is negative);
Ki-67 and mitotic count;
completeness of resection, distance of the surgical margins from the tumoral edge,
depth of invasion;
signs of malignancy (angiolymphatic and/or perineural invasion, necrosis, infiltration
of the capsule and/or of gastrointestinal (GI) wall and/or surrounding tissues);
number of examined lymph nodes, and number of lymph node metastases; presence of micrometastases;
diameter of largest metastasis;
presence of distant metastases, if demonstrated;
functional activity (if appropriate).
The report should be concluded with the WHO diagnosis and classification of the lesion
(NET G1–G2 or NEC G3) based on proliferative index (Ki-67 and/or mitotic count), and
with the tumor stage (the staging system should be specified).
The minimum pathology data set for resected specimens (both primary and metastatic)
should include [37]:
site;
diagnosis (e.g., pure neuroendocrine neoplasm);
differentiation (i.e., well or poor);
proliferation (i.e., G1 or G2 or G3).
Genetic assessment
Approximately 5–10 % of GEP-NENs have a hereditary background as part of tumor susceptibility
syndromes: multiple endocrine neoplasia type 1 (MEN-1), von Hippel-Lindau disease
(VHL), neurofibromatosis type 1 (von Recklinghausen disease, NF1) and the tuberous
sclerosis complex (TSC). All are inherited autosomal dominant disorders [38].
MEN-1 GEP-NENs are the second most common manifestation of MEN-1, reported in 30–70 %
of cases in different series [mostly non-functioning (NF)] [39, 40]. A germ-line MEN-1
mutation is identifiable in about 80–90 % of familial cases [41] and in about 42 %
of sporadic cases [42]. Germline mutations arise de novo without any family history
in approximately 10 % of patients [43]. MEN-1 mutation testing should be offered to
index cases and to their first-degree relatives, even if asymptomatic [40]. Genetic
counseling is recommended [40]. The family members who carry the MEN-1 mutation require
routine surveillance for early detection of endocrine tumors, whereas those who do
not carry the mutation can be reassured. When molecular genetic testing is not available
locally, patients highly suspected for MEN-1 should be addressed to a referral centers.
No genotype/phenotype correlations have been demonstrated in MEN-1 syndrome [44, 45].
VHL Endocrine pancreatic NF tumors occur in 11–17 % of patients with VHL disease [46].
The penetrance of VHL mutations is almost complete by age 65 years [47]. Genetic testing
detects mutations in virtually all affected individuals [48] and should be offered
to all individuals with clinical evidence of VHL and to first-degree relatives. As
ophthalmologic screening for those at risk for VHL disease begins before age five,
molecular genetic testing is suggested also in young asymptomatic children [49, 50].
NF1 GEP-NENs occur in 1 % of the NF1 patients [51]. Half of affected individuals have
NF1 as the result of a de novo mutation. The offspring of an affected individual is
at a 50 % risk of inheriting the altered NF1 gene, and the disease manifestations
are extremely variable, even within the same family [52]. Molecular testing for NF1
is not usually recommended in the clinical practice: screening for NF1 mutations is
useful only in individuals who do not completely fulfill the NIH diagnostic criteria.
TSC A few cases of pancreatic (p)NENs have been described in patients with TSC [53–55].
The diagnosis of TSC is usually based on clinical findings and mutations can be identified
in approximately 85 % of individuals who meet the diagnostic criteria [56]. Two-thirds
of affected individuals have TSC as the result of a de novo mutation.
Laboratory assessment
The determination of GEP-NENs serum markers should not be used as a first-line diagnostic
tool whereas it is appropriate for monitoring the response to treatment and for long-term
follow-up [57, 58].
Serum markers should be determined after:
an established diagnosis or strong clinical suspicion of GEP-NEN;
exclusion of physiologic and pathologic confounding conditions.
NEN markers may be regarded as “unspecific” or “disease-specific”.
“Unspecific markers”
Chromogranin A
Chromogranin A is a widely employed serum marker for GEP-NENs, but its use presents
limitations [59]. CgA circulates under different antigenic forms and no universal
calibration standard is available [60]. IRMA and RIA results may be considered roughly
equivalent [61], but the reference intervals are variable and results obtained with
different assays cannot be compared.
Chromogranin A level may be increased in a number of pathologic conditions (Table 3),
and in healthy subjects after eating or physical exercise. Accordingly, CgA levels
are highly variable in the general population [62], and may partially overlap between
GEP-NEN patients and controls. Hence, CgA has a poor first-line diagnostic value [5,
60, 62–66].
Table 3
Potential confounders causing CgA increase [64]
Neoplastic (other than GEP-NENs)
Breast cancer Prostate cancer Ovarian cancer Hepatocarcinoma
Pancreas adenocarcinoma Colon cancer
Non-neoplastic
Kidney or heart failure Endocrine diseases (hyperthyroidism, hyperparathyroidism) Local
or systemic inflammatory disease Chronic obstructive broncho-pulmonary disease Gastro-enteric
pathologies: chronic atrophic gastritis, pancreatitis, inflammatory bowel disease,
cirrhosis, chronic hepatitis
Proton pump inhibitors (PPIs) increase (up to sevenfold) CgA levels. The effects of
PPIs persist for several days after drug discontinuation. Therefore, CgA testing should
be performed after an at least 2-week PPIs withdrawal [62, 67]. The effects of H2-receptor
antagonists (H2RAs) on CgA are still controversial [68].
Diagnostic accuracy of CgA depends on different variables:
tumor burden (sensitivity 60–100 vs. 29–50 % in metastatic and localized disease,
respectively) [62, 64, 69];
type and site of tumor (sensitivity 96 vs. 75 % in functioning and NF tumors, respectively)
[63, 70].
Other unspecific markers
Neuron-specific enolase (NSE) is an enzyme found in neuroectoderm-derived cells. The
presence of NSE has been reported in thyroid and prostate carcinomas, neuroblastomas,
small-cell lung carcinomas, and pheochromocytomas. The clinical usefulness of this
marker is hampered by its poor specificity [71]. NSE level is elevated in 30–50 %
of patients with NEN, particularly those with poor differentiation. The combined determination
of NSE and CgA may improve sensitivity in GEP-NEN diagnosis [72].
Pancreatic polypeptide (PP) is secreted by specialized pancreatic islet cells and
inhibits gut motility and pancreatic exocrine secretion. PP has been proposed for
the diagnosis and monitoring of NF pNENs, as its combination with CgA increases sensitivity
up to 93 % [69]. Its routine use is not recommended due to the low diagnostic performance
(sensitivity 63 % and specificity 81 %). PP levels may increase in old age, diarrhea,
laxative abuse, gut inflammatory processes and chronic renal disease.
Beta subunit of human chorionic gonadotropin (hCG), a glycoprotein synthesized by
the syncytiotrophoblastic cells of the placenta during pregnancy, may be increased
in patients with pNENs [73], but has no use in every day practice.
As a whole, the clinical usefulness of the above reported markers is limited.
“Specific markers”
5-HIAA
5-HIAA, the main urinary metabolite of human serotonin, is determined by HPLC on 24 h
urine samples. Results may be expressed as absolute values or as a ratio to creatinine
excretion.
Some pre-analytical variables, mostly tryptophan/serotonin-rich foods and drugs, may
interfere with serotonin metabolism (Table 4) [74]. These products should be avoided
prior to urine collection, respectively, for at least 72 and 24 h [64].
Table 4
Drugs and foods interfering with 5-HIAA assay
False negative results
Acetylsalicylic acid Phenothiazines: chlorpromazine, promethazine Imipramine and
MAO-inhibitors ACTH Ethanol MethylDOPA and hydrazine derivatives Ketoacids LevoDOPA Isoniazid,
methenamine, gentisic and homogentisic acid Streptozotocin Heparin
False positive results
Acetaminophen, naproxen, phenacetin Caffeine, nicotine Coumaric acid Diazepam Ephedrine Fluorouracil,
melphalan Phenobarbital Phentolamine, reserpine, guaifenesin, mephenesin Methamphetamine,
Phenmetrazine Methocarbamol Mesalamine Foods: bananas, avocados, kiwi, pineapples,
peanuts, tomatoes, plums, eggplants, walnuts, pecans, coffee, tea, cocoa/chocolate,
vanilla, sweets, and cookies (sugar and marmalade are allowed)
The normal 5-HIAA urinary excretion ranges from 2 to 8 mg/day, but unspecific elevations
(up to 30 mg/day) may be found in malabsorption syndromes, such as celiac and Whipple’s
disease [75–77].
The determination of 24-h urinary excretion of 5HIAA has a sensitivity of over 90 %
and a specificity of 90 % for full-blown carcinoid syndrome (CS, see “Box 2, Carcinoid
syndrome”). Urinary 5-HIAA excretion in these patients is reportedly higher than 90 mg/day
(up to 2,000 mg/day). The test sensitivity, however, is definitely lower in absence
of clinical symptoms [77, 78].
There is a possibility to analyze plasma 5-HIAA which might replace urinary-5-HIAA
in a future [79].
Various blood serotonin assays have been proposed, but their actual accuracy has not
been established. False positives may occur due to several interfering factors, as
the release of platelet serotonin or the previous ingestion of tryptophan/serotonin-rich
foods [80]. Accordingly, serotonin determination is not recommended in clinical practice.
Gastrin
Gastrin determination has a key role in the evaluation of patients with signs and
symptoms suggestive of Zollinger–Ellison syndrome (ZES, see “Box 3, Gastrinoma”).
Various antigenic isoforms of gastrin circulate in the blood. Care must be taken because
some commercial immunoassay kits detect only the gastrin-17 molecule [81] and may
cause false positive results.
Hypergastrinemia is commonly defined as a fasting serum gastrin above 100 pg/mL. Simultaneous
measurement of gastric pH on a single sample is needed to rule out secondary hypergastrinemia
due to other causes. In achlorhydria, pernicious anemia or atrophic gastritis high
gastrin levels are usually associated to high (i.e., >4) pH values. On the contrary,
serum gastrin levels >1,000 pg/mL combined with a <2 gastric pH are virtually diagnostic
of ZES. Falsely elevated gastrin levels may be due to a few drugs (Table 5) that should
be discontinued at least 2 weeks before the test [77, 82–86].
Table 5
Main drugs and foods that may interfere in gastrin assay
False negative results
Acetylsalicylic acid LevoDOPA
False positive results
Hypochlorhydria/achlorhydria due to chronic use of PPIs and H2RAs or chronic atrophic
gastritis (often associated with pernicious anemia) Helicobacter pylori infection Gastric
outlet obstruction Renal failure Antral G-cell syndromes Short-bowel syndrome Retained
antrum
In general, gastrin levels are higher in pancreatic than in duodenal NENs, and are
proportional to tumor burden and in patients with metastatic disease, exceedingly
high gastrin levels may be observed. However, the majority of patients with ZES show
mildly elevated (e.g., 150–1,000 pg/mL) gastrin levels, partially overlapping those
of patients with renal insufficiency, small-bowel resection, retained gastric antrum,
or on potent antisecretory drugs [87]. When the diagnosis is equivocal, a secretin
stimulation test is needed. A gastrin increase >120 pg/mL over basal level is considered
diagnostic [88].
Insulin
The occurrence of repeated symptomatic hypoglycemia (<60 mg/dL) is suspicious for
insulinoma (see “Box 4”) in subjects without diabetes. The diagnosis is confirmed
by the presence of non-suppressed insulin levels in presence of low glucose levels
(see “Spontaneous hypoglycemia”). To rule out a spurious hypoglycemia, laboratory
processing should not be delayed. In subjects with leucocytosis glucose determination
should be repeated with a collection tube that contains an inhibitor of glycolysis.
In presence of an episode of spontaneous severe hypoglycemia with hyperinsulinism,
the simultaneous measurement of serum C-peptide and beta-hydroxybutyrate is appropriate.
If factitious hypoglycemia is suspected, urinary sulfonylureas should be tested as
well. The work-up may be completed with the measurement of serum proinsulin [89].
This test, even if not widely available, is diagnostic of insulinomas secreting immature
forms of insulin.
In selected patients with endogenous hyperinsulinism, autoimmune hypoglycemia, suspected
on the basis of negative imaging tests and coexistence of autoimmune disorders, should
be ruled out with the determination of insulin autoantibodies [90].
If the patient is not hypoglycemic when observed, the association of severe hypoglycemia
with non-suppressed insulin levels should be seeked under the conditions in which
hypoglycemia would be expected (see provocative testing, “Spontaneous hypoglycemia”)
[89].
Other specific markers
Glucagon: Glucagonoma is associated with serum glucagon concentrations higher than
500 pg/mL and a characteristic clinical syndrome (diabetes mellitus and cutaneous
manifestations, such as migratory necrolytic erythema, nail dystrophies, stomatitis,
etc.) [91]. Glucagon concentrations higher than 1,000 pg/mL are virtually diagnostic
for the disease, but some patients may exhibit levels within the physiologically elevated
range. Moderate elevations in serum glucagon may be caused by protracted fasting in
normal subjects or by renal and hepatic failure, trauma, sepsis, pancreatitis, abdominal
surgery, and Cushing’s syndrome.
Due to the fast degradation of glucagon in vitro, blood must be collected in test
tubes containing aprotinin and should be rapidly delivered to the laboratory. Results
obtained with different glucagon assays may profoundly differ, due to the different
calibration standards and the variable cross-reactivity with glucagon isoforms.
Vasointestinal peptide (VIP): Vasointestinal peptide-secreting tumors cause the Verner–Morrison
syndrome, characterized by variable combination of watery diarrhea (>700 mL/day even
during fasting, with tea-colored, odorless stools), hypokalemia, achlorhydria, weight
loss, metabolic acidosis, hypercalcemia, glucose intolerance, and flushing. The diagnosis
is established by high-volume secretory diarrhea associated with VIP levels higher
than 75 pg/mL (to be confirmed by a second RIA determination) [92, 93]. VIP blood
concentration is, in fact, extremely low in healthy subjects. Commercial kits are
available, but their use is usually limited to tertiary referral centers.
Imaging procedures
Radiologic procedures
Ultrasonography
Transabdominal US is an inexpensive, safe, rapid and non-invasive tool. US accuracy
is, however, operator dependent and its sensitivity is generally low (13–27 %), when
compared with MultiDetector CT (MDCT) and magnetic resonance imaging (MRI) [94]. In
case of pNEN, a mean 39 % US detection rate has been reported [95, 96].
Contrast-enhanced US (CEUS) enables identification of hypervascular lesions, even
in case of fast-flow tumor circulation, as in NF pNENs. Therefore, CEUS is significantly
superior to B-mode US both in the detection of NF pNENs and in the diagnosis of liver
metastases, visualized as hyperenhancing non-homogeneous lesions [96–98], with a reported
sensitivity of 82 % [99, 100]. US may help in defining complications of advanced disease
(i.e., biliary stricture) and/or guide diagnostic or therapeutic procedures [101].
Endoscopic ultrasonography and EUS-guided FNA, a fundamental procedure for the diagnosis
of pNENs [96, 102, 103], will be treated in “Pancreatic NENs”.
Multislice triple phase CT
Multidetector CT is considered the first choice imaging modality for detection, staging
and follow-up of GEP-NENs. When compared to conventional CT, MDCT allows a markedly
higher spatial and temporal resolution. MDCT sensitivity and specificity are increased
due to multiphase scanning. Images should be acquired in precontrast, arterial, portal
and equilibrium phases.
Non-functioning pNENs and NEN liver metastases typically appear as hypervascular lesions.
In the evaluation of NF pNENs, the combination of arterial dominant-phase and portal
venous-phase CT improves the detection of primary tumors and hepatic metastases [96].
Reported mean sensitivity and specificity of MDCT are 73 % (63–82 %) and 96 % (83–100 %)
for pNENs, and 82 % (78–100 %) and 92 % (83–100 %) for liver metastases, respectively
[104–106].
When a small ileum lesion is suspected, MDCT enterography can be performed by distending
the small bowel with a large volume of neutral or low-attenuating oral contrast medium
[107–109]. The reported sensitivity and specificity of MDCT enterography are variable,
ranging from 50 to 85 % and from 25 to 97 %, respectively.
Due to radiation exposure, MDCT examination should be tailored, particularly in young
people, to reduce the scanned volume and the number of phases.
MRI
Like MDCT, MRI offers a high spatial and time resolution with the possibility of multiplanar
acquisition and reconstruction and multiphase examination after contrast injection.
Along with the absence of ionizing radiations, an advantage of MRI over MDCT is the
intrinsic signal difference (contrast) between the neoplasm and the healthy parenchyma.
This characteristic is increased with imaging sequences based on proton diffusion.
If compared with MDCT, the major drawbacks of MRI are the higher cost, lower accessibility
and longer scanning time. Furthermore, MRI is more dependent on patient cooperation.
At MRI, GEP-NENs show the same enhancement characteristic described for MDCT. As for
contrast medium, Gadolinium-based (Gd-EOB DTPA) agents (Primovist for MRI) should
not be used in patients with advanced renal function impairment.
Magnetic resonance imaging demonstrates a particular sensitivity for liver, bone,
soft-tissue, and central nervous system metastases [87, 95]. Multiphase CT scan and
MRI have similar effectiveness in the detection of islet cell tumors if fat-saturated
T1-weighted and delayed enhanced T1-weighted sequences are included.
In clinical practice, MRI should be used when MDCT does not offer clear-cut results
or when contrast medium is contraindicated [95]. Due to the absence of radiation exposure,
MRI is used, in association with US, either as a screening image modality in young
patients or in long-term surveillance [110].
Nuclear medicine procedures
SSTR functional imaging
Up to 80 % of GEP-NENs express primarily SSTR2 and SSTR5: this feature enables imaging
with SA compounds, labeled with radioactive tracers.
The most common radiopharmaceutical SA is 111In-pentetreotide (commercially available
as Octreoscan®) used for scintigraphy, SPECT and SPECT/CT [111, 112]. Modern hybrid
acquisition systems as SPECT/CT allow a coregistration of functional and morphologic
imaging, which improves the localization of lesions [113].
Due to its high affinity to SSTR2, Octreoscan® shows a higher detection rate of NEN
lesions as compared to conventional imaging, with a sensitivity ranging from 67 to
near 100 % [114–119].
Among other radiolabeled SA, 68Ga-DOTA-D-Phe1-Tyr3-octreotide (DOTATOC) binds SSTR2
and SSTR5 with higher affinity than Octreoscan® [120]. In light of higher spatial
resolution (3–5 mm) and better quantification of tracer uptake offered by PET in comparison
with scintigraphy, PET and PET/CT scan with 68Ga-DOTATOC have significant advantages
over SRS imaging, particularly in organs with high physiologic uptake (e.g., liver)
and in case of small lesions (<1.5 cm) [121–123]. Furthermore, 68Ga-DOTATOC has proven
to be superior to CT and bone scintigraphy in the detection of bone metastases from
GEP-NENs [124].
Similar results have been obtained with PET imaging using other 68Ga-labeled peptides
(e.g., 68Ga-DOTATATE and 68Ga-DOTANOC) [125–130]. PET/CT with 68Ga-labeled SA is quite
effective, both in terms of diagnostic accuracy and impact on clinical management
[131–134]. Accordingly, this imaging procedure is recommended for routine use [73].
PET/CT with 68Ga-labeled SA is presently available at a limited number of institutions,
but will hopefully become diffusely adopted worldwide in the next future (Table 6).
Table 6
Comparison between Octreoscan and Ga-DOTA-peptides
Availability
Duration
Accuracy
NPV
PPV
111In-pentetreotide (Octreoscan®)
Widespread
2 days
++
++
+++
68Ga-DOTA-conjugate peptides
Low
2 h
+++
+++
+++
Clinical indications for nuclear imaging based on radiolabeled SA are [135]:
primary tumor localization and staging;
restaging (detection of residual, recurrent or progressive disease);
SSTR status evaluation (patients with high positivity are more likely to respond to
octreotide therapy);
response to therapy monitoring;
selection of patients eligible for peptide receptor radionuclide therapy.
As octreotide therapy can theoretically interfere with 111In-pentetreotide uptake,
a brief (1–2 months) withdrawal of long-acting SA or a transient switch to short-acting
SA should be considered [135].
PET with other tracers
18F-FDG-PET/CT has been traditionally thought to play a minor role in GEP-NENs imaging
due to the expected low FDG uptake of low-grade GEP-NENs [136]. As FDG uptake is greater
in high-grade tumors, 18F-FDG-PET/CT has been proposed in patients with advanced,
metastatic GEP-NENs with promising results [137, 138]. In addition, combined functional
imaging with both 68Ga-DOTATATE and 18F-FDG may be useful for a more comprehensive
tumor assessment in intermediate and high-grade tumors [125]. Two recent studies confirm
that FDG-PET is a sensitive technique for staging GEP-NENs with high (≥10–15 %) Ki-67
[139, 140]. As for other tumors, it has been suggested that FDG positivity points
to a worse prognosis [141–143].
18F- and 11C-labeled amine precursors l-dihydroxyphenylalanine (DOPA) [144–148] and
5-hydroxy-l-tryptophan [146, 149, 150] have been utilized for PET imaging of GEP-NENs
in a limited number of studies with promising results. A still investigational tool
is 18F-fluorothymidine PET that seems to provide non-invasive assessment of cell proliferation.
Finally, there is the possibility of utilizing glucagon-like peptide-1 receptor imaging
for the localization of insulinomas [151]. Clinical application of these radiopharmaceuticals
is not for routine use and needs confirmation.
Endoscopic procedures
Upper and lower gastrointestinal NENs
Upper gastrointestinal endoscopy (EGDS) with gastric biopsy is required for the detection
of gastric NENs.
Esophago-gastro-duodenoscopy is the only recommended imaging procedure in small (<1 cm)
enterochromaffin-like cell tumors (ECLomas). Type 1 and 2 gastric NENs generally present
(in 65–77 % of cases) as small (<2 cm) multifocal polypoid mucosal protrusions in
the body and/or fundus of the stomach. Type 3 tumors are usually solitary, ulcerated
and larger than 2 cm. In addition to biopsies of the largest polyps, samples should
be taken from the antrum (two biopsies) and body/fundus (four biopsies) [152, 153].
Regardless of the type of gastric NEN, EUS may help to determine the presence of tumor
invasion of the gastric wall and it is recommended before the resection of polyps >1–2 cm
in diameter. EUS is useful for the assessment of the regional lymph node involvement
and for cyto-histologic confirmation by FNA [154].
Duodenal NENs are approached in the same manner, namely EGDS with biopsies and EUS
[155, 156].
The majority of rectal NENs are diagnosed endoscopically. Most lesions present as
polyps, which are completely removed by snare polypectomy, but their diagnosis may
be established only after histologic evaluation. Full colonoscopic assessment is required
to exclude concomitant colonic disease as part of staging, and the possibility of
synchronous carcinoma must be excluded. EUS is very useful in assessing rectal NENs
extension preoperatively and it accurately assesses tumor size, depth of invasion
and perirectal lymph node metastases. Hence, EUS provides information critical for
the choice of final treatment (endoscopic vs. surgical) [157, 158].
Small-bowel NENs
Direct visualization of small-bowel NENs may be obtained by standard colonoscopy if
the tumor is prolapsed through the ileocecal valve into the colon, or if intubation
of the ileum via the ileocecal valve is performed. Newer modalities to investigate
the proximal parts of the ileum or the jejunum include video-capsule endoscopy (VCE)
and enteroscopy. Small-scale studies have reported successful detection of occult
small-bowel NENs by VCE where other techniques have failed. It is advisable to use
a dissolvable “patency” capsule to avoid capsule “retention” within strictures. Major
VCE limitations are as follows: (a) precise localization of the tumor is not usually
possible; (b) in case of predominantly extraluminal GEP-NEN, the evaluation of the
tumor cannot be accurate; and (c) cost and operating time. VCE revealed a sensitivity
of 60 % and a specificity of 100 % as compared to CT enteroclysis [107, 159].
In selected cases, double balloon enteroscopy (DBE) seems to be a valuable method.
It allows histologic confirmation by luminal biopsy and accurate preoperative localization
by tumor marking with ink injection. A 33 % diagnostic yield of DBE for primary tumor
detection in patients with metastatic or suspected GEP-NEN has been reported [160].
Pancreatic NENs
Endoscopic ultrasonography is an effective tool to identify pNENs, which typically
appear as well-defined hypoechoic, hypervascular masses. Cystic change, calcifications,
and necrosis are common in large tumors. EUS-guided FNA (or biopsy, FNAB) is useful
to confirm the diagnosis of pNEN. EUS sensitivity is quite high (79–100 %) with a
PPV close to 100 % [161–164]. The accuracy decreases in case of lesions located in
the pancreatic tail [165]. While EUS shows a higher sensitivity than cross sectional
imaging in the diagnosis of small, multiple pNENs in MEN-1 or VHL syndromes, its accuracy
in the detection of small duodenal tumor is controversial. The combination of dual-phase
thin-section multidetector CT and EUS has been reported as the most accurate procedure
to detect insulinomas [166]. EUS plus FNA is highly cost-effective when used early
in the preoperative work-up, reducing the need for additional invasive tests [167,
168]; complication rate is quite low (<1 %) [168]. A close correlation between aspiration
cytology and the final histology after resection has been demonstrated [169]. EUS
is thus useful in the preoperative setting as it provides information that significantly
influences the therapeutic planning [170].
A step-by-step multidisciplinary approach to clinical diagnosis
The suspicion of GEP-NEN can be raised in four different scenarios: (1) incidental
finding either in a totally asymptomatic patient or in a patient with symptoms unrelated
to GEP-NEN; (2) symptomatic patient with GEP-NEN-related local effects, (3) syndromes,
and (4) metastases from unknown primary GEP-NEN. The first two scenarios are typical
of NF GEP-NENs.
Incidental finding
GEP-NENs are often suspected following incidental imaging (e.g., US, CT, MRI) or endoscopic
findings, in patients without signs or symptoms related to GEP-NEN [1, 3, 6].
The patient should be checked for minor GI complains (diarrhea, constipation, peptic
disease, gastroesophageal reflux), any palpable mass, skin and metabolic signs/symptoms,
possibly suggesting a functioning syndrome. An accurate clinical history of the patient’s
family should also be collected to confirm or rule out a hereditary syndrome [6].
GEP-NENs suspected at endoscopy
Incidental diagnosis of GEP-NENs often follows the histologic examination of polypoid
lesions found during endoscopic procedures in an asymptomatic patient. Otherwise,
gastroduodenal and colorectal NENs may be suspected in case of single or multifocal
polypoid mucosal protrusions [152, 155, 158], even though no endoscopic finding is
highly specific of NEN.
An endoscopic biopsy of the suspected lesion is mandatory. In case, the endoscopic
biopsy is either not feasible or non-diagnostic, morphologic imaging studies should
be programmed as the second step. Image-guided or laparoscopic biopsy should be discussed
by the multidisciplinary team. Functional imaging could subsequently be performed
as a complementary staging-prognostic tool.
No lab tests are indicated in the diagnostic work-up. The finding of hypergastrinemia,
achlorhydria, macrocytic anemia, B12 deficiency and/or intrinsic factor antibodies
may be useful to categorize a gastric NEN (Fig. 2).
Fig. 2
Diagnostic flow-chart for GEP-NEN suspected at endoscopy
GEP-NEN suspected at morphological (US/CT/MR) imaging
This incidental finding is usually related to primary pancreatic tumor or liver metastases
from a GEP-NEN.
A pNEN might be suspected in case of hypoechoic, hypervascular, and/or well-defined
lesions at US/CEUS and of enhancing hypervascular lesions at CT scan or MRI. Cystic
changes, calcifications, and necrosis are frequently observed in large lesions [171].
False positives, especially in case of US imaging, like hemangiomas, hepatocellular
and pancreatic carcinomas, intraductal pancreatic mucinous tumors, adenomas and metastasis
from other tumors [94, 95, 97–101] should be ruled out by the multidisciplinary team.
A histologic/cytological specimen should possibly be obtained [96, 172].
Once the diagnosis of GEP-NEN is pathologically confirmed, proceed to morphologic
and functional staging (see below, “When and how to stage a previously diagnosed GEP-NEN”).
If biopsy is unfeasible or inconclusive, a second imaging technique (e.g., EUS, CEUS,
liver-specific contrast-enhanced MRI, etc.) should be performed according to local
expertise and availability [6].
Metastatic lesion(s) from occult primary may require a specific work-up (see below,
“Work-up in the patient with metastatic disease and unknown primary tumor”).
No lab tests are recommended in the diagnostic work-up. Nevertheless, elevated 5-HIAA
urinary excretion is highly specific of GEP-NEN liver metastases and may, therefore,
be a strong diagnostic clue in case of a non-diagnostic biopsy. In patients with pNENs,
the occurrence of subclinical, vague functional signs/symptoms possibly indicating
a functional syndrome should always be carefully checked. Accordingly, specific hormonal
assays may be required in selected cases (Fig. 3).
Fig. 3
Diagnostic flow-chart for GEP-NEN suspected at morphological imaging
GEP-NEN suspected after elevated serum CgA levels
Chromogranin A must never be considered a first-line diagnostic test. Nevertheless,
NEN suspicion may occasionally be driven by the finding of elevated serum CgA levels,
measured on the basis of unspecific symptoms or signs.
Before proceeding to imaging/endoscopic studies, all factors affecting CgA levels
must thoroughly be ruled out (see “Table 3”). A second CgA determination is always
required for confirmation. In patients on PPI treatment, serum CgA should be repeated
after a two-week PPI withdrawal.
If CgA levels are confirmed elevated in absence of confounding factors, transabdominal
US should be performed. A further diagnostic work-up should be discussed by a multidisciplinary
team or a referral center should be involved (Fig. 4).
Fig. 4
Diagnostic flow-chart for NEN suspected after high CgA
Symptomatic patient with symptoms due to GEP-NEN-related local effects
When to suspect a GEP-NEN
Non-functioning GEP-NENs (Box 1) may become symptomatic when they compress or invade
adjacent structures or when they metastasize. The suspicion of GEP-NEN might be raised
by suggestive imaging findings (see above) and/or by the apparently slow progression
of the disease [73]. Lab findings (e.g., frankly elevated CgA levels in absence of
confounding factors) may reinforce the suspicion. As previously stated, only pathology
(cytological or histologic characterization), however, will establish the diagnosis
[6].
Box 1
Non-functioning GEP-NENs
Definition:
NF GEP-NENs are tumors that do not show symptoms related to hormonal hypersecretion.
Intracellular hormones or peptides may be demonstrated by IHC, but they are either
not secreted, or secreted in quantities unable to elicit a clinical syndrome and/or
in an inactive form [
3
]. Clinical presentation of NF GEP-NENs depends upon the site of origin and metastases.
They can be incidentally discovered when asymptomatic due to the widespread use of
diagnostic imaging [
1
,
3
]. Clinical presentations according to the site of origin are listed below.
Pancreas:
Up to 60% of pNENs is NF. Most NF pNENs are well differentiated. Annual incidence
is 1.8 and 2.6 per million in females and males, respectively [
3
]. NF pNEN were traditionally diagnosed late in the course of the disease, with metastases
in 46 to 73% of cases, but presently the number of incidentally found small lesions
is steeply increasing. Presenting symptoms and signs are abdominal pain (35–78%),
weight loss (20–35%), anorexia and nausea (45%), intra-abdominal hemorrhage (4–20%),
jaundice (17–50%), and a palpable mass (7–40%) [
96
,
172
]. NF pNEN may occur in familiar syndromes such as MEN-1, VHL, and TSC.
Gastrointestinal:
NENs are frequently detected during a screening program or an imaging exam performed
to search the primary tumor in an asymptomatic but metastatic patient [
1
,
3
]. Alternatively, a common clinical presentation is abdominal pain that may be caused
by gastro-intestinal dysmotility or obstruction (associated or not to nausea, vomiting
or constipation), or by bacterial overgrowth. Less common symptoms and signs are jaundice,
weight loss, fatigue, fever and bleeding (massive or dripping). Clinical presentation
of appendiceal NEN may mimic acute appendicitis [
1
,
3
]. Obstructive symptoms are typical of small bowel, whereas minor bleeding is frequent
in rectal disease [
6
,
73
,
173
].
Work-up in the patient with local compressive symptoms
A detailed history and complete physical examination are required.
Abdominal pain is the most common presenting symptom of NF GEP-NENs and may be related
to the primary tumor or metastatic lesions [1, 3]. Pain localization and characteristics
should be carefully examined. Four different scenarios can be distinguished.
Isolated abdominal pain
A persistent and oppressive upper-abdominal pain may signal a pancreatic or retroperitoneal
mass (pattern 1a) [96, 172], while a discontinuous cramping pain usually refers to
an intestinal origin (pattern 1b) [73, 173]. In the former case, a radiological imaging
should be performed first, followed by endoscopy/EUS as second step for pancreatic
and duodenal lesions. In the latter case, endoscopy is recommended [73, 173]. A cytologic/histologic
sampling should be obtained whenever possible (Fig. 5) [96, 172].
Fig. 5
Diagnostic flow-chart for GEP-NEN suspected after pattern 1a and 1b
An ill-defined and diffuse abdominal pain (pattern 1c) can also be related to liver
or nodal metastases. Abdominal US followed by a whole-body CT scan and a US-guided
biopsy should be performed (Fig. 5).
Subocclusive picture
It may be due to a large, often metastatic, ileal NEN and/or peritoneal carcinomatosis.
Depending on the severity of the clinical picture, a direct abdomen-X-ray and/or an
endoscopy could be performed [73, 173]. If an extrinsic obstruction is suspected,
then an abdomen CT scan should be performed. If a peritoneal carcinomatosis is suspected,
a transit evaluation water-soluble contrast medium X-ray could be useful (Fig. 6).
If possible, histological specimens should be obtained through endoscopy. If not,
a US/CT-guided biopsy of the liver or other site lesions or laparoscopy-guided biopsy
should be discussed in a multidisciplinary team.
Fig. 6
Diagnostic flow-chart for GEP-NEN suspected after subocclusive picture
Jaundice
This clinical presentation points to the involvement of the liver, biliary tract or
pancreas. Liver function and structure should be assessed by blood tests and US, to
rule out the obstruction of the biliary tract. Compressive effects of lymphadenopathies
or a pancreatic mass may cause an extra-hepatic tract dilatation, whereas liver metastases
are more likely related to an intra-hepatic tract dilatation [96, 172]. In case of
obstructive jaundice, a cholangio-MRI and endoscopic-retrograde-cholangio-pancreatography
(ERCP) can be considered. Cytology by means of brushing or histology can be obtained
through ERCP. Whole-body CT scan and endoscopy should be used to define the primary
site of the tumor and for staging purpose (Fig. 7).
Fig. 7
Diagnostic flow-chart for GEP-NEN suspected after jaundice
Gastrointestinal bleeding
It can be related to the compressive and infiltrating effects of a tumor mass. Bleeding
can be massive (hematemesis, melena and rectal bleeding) or dripping and occult. Blood
tests, iron assessment and endoscopy must be performed. Massive bleeding always requires
hospitalization and may require angiography [73, 173]. In case of lesions located
in the stomach-duodenum or in terminal ileum-colon tract, a histologic diagnosis may
be obtained through biopsy during EGDS or ileo-colonoscopy. If upper and lower endoscopy
is negative, enteroscopy, enteroCT/MRI, VCE should be discussed in the multidisciplinary
team according to the local availability and expertise (Fig. 8). For lesions located
in the small bowel, a surgical diagnostic/therapeutic approach should be considered
Fig. 8
Diagnostic flow-chart for GEP-NEN suspected after GI bleeding
Symptomatic patient with syndromes
Diarrhea and flushing
Clinical approach: when to suspect a GEP-NEN
The patient with diarrhea and flushing should raise the suspicion of CS (Box 2).
Carcinoid syndrome diagnosis may be difficult. A detailed history and complete physical
examination are must. Symptoms may be under-reported by patients or be attributed
to other, more common GI disorders. Differential diagnoses include irritable/inflammatory
bowel diseases, microscopic colitis, food intolerance/allergy, bacterial overgrowth,
celiac disease, hypersecretory states (i.e., gastrinoma, see “Resistant/relapsing
ulcer disease”), chronic pancreatitis, other neoplastic (i.e., colon carcinoma, lymphoma)
and non-neoplastic conditions (asthma, anxiety, alcoholism) [6].
Diarrhea in patients with CS is chronic, predominantly secretory, does not change
with fasting, and is associated with fluid and electrolyte imbalance. A detailed history
of the diarrhea and specific questioning about other possible manifestations of CS
(i.e., facial flushing) are required. The stools are usually watery and result from
intestinal hypermotility and hypersecretion. Nocturnal diarrhea is generally considered
as characteristic of CS. The incomplete response to antidiarrhoic treatment should
raise the suspicion of possible CS [174].
Flushing is the most common symptom in CS. Eating, emotion, alcohol, and exercise
may worsen flushing. The face, neck and upper trunk usually turn pink to red in color
and the skin is characteristically dry. Flushing may also be associated with transient
hypotension and bronchoconstriction. Other causes of flushing/sweating disorders to
be considered are [175]:
pheochromocytoma, menopause, ZES, and medullary thyroid carcinoma (intermittent flushing);
alcoholism, polycythemia, mitral stenosis, and Cushing’s syndrome (constant flushing).
Box 2
Carcinoid syndrome
CS is classified as typical or atypical, accounting for 95% and 5% of total cases,
respectively [
176
,
177
,
178
].
Typical CS occurs in about 15-20 % of patients with jejuno-ileal NENs, with liver
metastases. In less than 5 %, it can be caused by retroperitoneal or ovarian metastases
that release serotonin or tachykinin, bypass the liver and enter the systemic circulation
[
179
,
180
,
181
,
182
]. These so-called “functioning carcinoids” exhibit a variable clinical presentation,
due to the type of secreted bioactive substances (serotonin, tachykinins, kallikreins,
and prostaglandins). Typical CS may present with cutaneous flushing (face, neck, upper
chest), GI hypermotility with pain (intermittent and crampy, described as dull, achy
and colicky, and not relieved by defecation), telangiectasia, peripheral edema, wheezing,
cyanosis, pellagra, and right-sided heart failure caused by cardiac valve abnormalities.
Symptoms may occur spontaneously or be triggered by alcohol intake, serotonin-rich
foods, and exercise [
182
].
Atypical CS is associated to overproduction of histamine and is characterized by prolonged
flushing, bronchoconstriction and hypotension [
178
]. Wheezing might suggest asthma that can be identified by lung function tests.
Carcinoid crisis is an extreme and life-threatening expression of the CS, induced
by the massive release of amines into the circulation following anesthesia, interventional
procedures or medication [
183
]. Main features of carcinoid crisis are: hypotension, rarely hypertension, tachycardia,
bronchial wheezing, and central nervous system dysfunction [
184
].
Carcinoid heart disease affects 10–20 % of the patients at presentation. CS causes
a thickening of the heart valves, impairing their proper function, resulting in insufficiency.
Heart failure typically involves the right-side valves. Signs and symptoms include
fatigue and shortness of breath during physical activity and peripheral edema in 1
out of 5 patients. Up to 50 % of deaths in CS are due to heart failure [
185
,
186
].
Work-up in the patient with suspected carcinoid syndrome
Before proceeding to the work-up, other causes of flushing with or without diarrhea
must be excluded (Table 7) [187]. To this aim it could be useful a 2 to 4-week detailed
self-recording of the flushing and diarrhea episodes.
Table 7
Differential diagnosis of flushing
Drugs
All vasodilators, calcium channel blockers, morphine and other opiates, etc.
Menopause
Associated with sweating
Mastocytosis
Flushing lasting longer than CS, may be accompanied by headache, dyspnea, palpitations,
abdominal pain and diarrhea
Medullary thyroid carcinoma
Associated with diarrhea in patients with advanced disease
Pheochromocytoma
Rare, but it may occur after a paroxysm of hypertension, tachycardia and palpitations
and is preceded by pallor
Since symptoms associated with CS can be triggered by alcohol intake and serotonin-rich
foods [188–190], the patient should follow an exclusion diet for at least 3 days before
starting urinary collection for 5-HIAA and should avoid for at least 24 h (or according
to half-life) drugs that affect this test (see Table 4).
Biochemical testing: Urinary excretion of 5-HIAA is the most useful test in patients
with typical CS due to jejuno-ileal NENs. Atypical CS is induced by gastroduodenal
and bronchial NENs that only rarely secrete serotonin because they lack DOPA-decarboxylase,
the enzyme that converts 5-hydroxytryptophan into serotonin [191]. These tumors may
thus produce 5-hydroxytryptophan and histamine instead of serotonin, but no assay
for urinary 5-hydroxytryptophan is commercially available, whereas histamine assays
are limited to very few centers.
5-HIAA testing is highly sensitive (up to 90 %) and specific (85–90 %) for the diagnosis
of CS. In patients with CS 5-HIAA levels are usually at least twice as high as the
upper normal limit. They may reflect the tumor burden and are rarely normal in patients
with CS [57, 73, 76, 77, 192–194]. Attention must always be paid to factors causing
falsely high or low levels (see Table 4).
Serum serotonin determination is not recommended because it may vary considerably
according to activity and stress levels [73]. CgA is poorly specific whereas NSE has
no diagnostic role [77, 193, 195].
Imaging procedures: Carcinoid syndrome is most frequently due to a NEN in the small
bowel associated with liver metastases [196]. Therefore liver assessment examinations
should be firstly performed, including US/CEUS (useful to drive biopsy), CT and MRI
(superior to CT for small lesions) [95, 197–200].
The type of work-up aimed to the detection of the primary tumor (see “Work-up in the
patient with metastatic disease and unknown primary tumor”) and to rule out atypical
situations should be discussed in a multidisciplinary panel, also taking into account
the possible surgical resection.
Functional imaging studies (SRS, or when available 68Ga-DOTA-peptide-PET) may help
in localizing the primary tumor and small metastases, and as a predictive factor for
somatostatin receptor driven therapies. Combination of SRS or PET with CT increases
the sensitivity [117].
In case of persistently negative results of morphological and functional studies,
the primary tumor may be located by intraoperative palpation [73].
Transthoracic echocardiography should be performed at diagnosis of CS and then annually
to detect any right-sided fibrosis involving tricuspid and pulmonary valves [201]
(Fig. 9)..
Fig. 9
Diagnostic flow-chart for suspected carcinoid syndrome
Resistant/relapsing ulcer disease
Clinical approach: when to suspect a GEP-NEN
ZES (Box 3) is characterized by gastric acid hypersecretion resulting in severe peptic
disease and gastroesophageal reflux disease (GERD) [202–205].
The majority of ZES patients presents with a single duodenal ulcer, peptic symptoms,
GERD symptoms or ulcer complications and diarrhea. Multiple ulcers or ulcers in unusual
locations are a less frequent presenting feature than in the past [8, 84, 85, 87,
202, 204–208]. With the widespread use of gastric antisecretory drugs, particularly
PPIs and H2RAs, symptoms may be masked. The diagnosis is most often suggested by a
long history of peptic ulcer disease or GERD symptoms or their recurrence after treatment
[83–85, 206, 208]. This delay may postpone the diagnosis of gastrinoma to a higher
stage of the disease.
Box 3
Gastrinoma
Gastrinoma is a functioning GEP-NEN, usually located in the duodenum or pancreas that
secretes gastrin and causes a clinical syndrome known as ZES.
The incidence of gastrinomas is 0.5–2/million population/year. Gastrinoma is one of
the most common functioning GEP-NEN in the general population [
8
] and occurs in 25–40 % of subjects with MEN-1 [
207
,
209
]. ZES occurs at an earlier age (mean 32–35 years] in patients with MEN-1 than in
those with sporadic disease [
204
,
207
,
209
].
Pancreatic gastrinomas may occur in any portion of the pancreas, while duodenal gastrinomas
are predominantly found in the first part of the duodenum including the bulb [
210
,
211
]. At surgery, 70–85 % of gastrinomas are found in the right upper quadrant (duodenal
and pancreatic head area), the so-called “gastrinoma triangle” [
210
,
211
,
212
].
The main symptoms classically associated to ZES are due to gastric acid hypersecretion
and are represented by abdominal pain (75–98 % of the cases), diarrhea (30–73 %),
heartburn (44–56 %), bleeding (44–75 %), nausea/vomiting (12–30 %), and weight loss
(7–53%) [
85
,
204
,
205
].
At presentation, >97 % of patients have an elevated fasting serum gastrin (FSG) level,
87–90 % have marked gastric acid hypersecretion (basal acid output >15 mEq/h) and
100 % have a gastric acid pH <2 [
202
,
213
].
The rate of malignancy is high with liver metastases in 30-40 % of cases [
214
].
Work-up in the patient with suspected gastrinoma
History and clinical examination are the first steps in the diagnosis of ZES. The
use of acetylsalicylic acid and other non-steroidal anti-inflammatory drugs, which
can mimic a ZES picture, should be ruled out [215].
Multiple endocrine neoplasms should be considered in all patients with ZES, especially
in case of familial or personal history of endocrine disease, kidney stones, other
NENs [88, 207]. Due to high penetrance of primary hyperparathyroidism in MEN-1 [40],
serum calcium and PTH are the first step to rule out the diagnosis.
Biochemical testing: Fasting serum gastrin is an excellent screening test (>98 % sensitivity).
False positive conditions should always be excluded (Table 5). The diagnosis of ZES
requires inappropriately elevated FSG levels in association with a >15 mEq/h (>5 mEq/h
in gastrectomized patients) basal acid output or in association with a gastric pH
<2.0. Under these conditions, FSG >1,000 pg/mL means a certain diagnosis of ZES. On
the contrary, a gastric pH >2.0 virtually excludes ZES [84]. In subjects under chronic
therapy with PPIs these drugs have to be withdrawn for at least 1 week [84, 216],
although the optimal wash-out time for PPIs should be longer (4 weeks). H2RAs exert
a less pronounced suppression of gastric acid output than PPIs [217, 218]. In case
of subjects on PPIs who are at risk of bleeding ulcer, diarrhea with dehydration or
hypokalemia, these drugs may be replaced with H2RAs for at least 1 week under medical
supervision [219, 220].
Secretin test (2 U/kg rapid infusion), a gastrin provocative test, may be performed
in controversial cases [77, 221]. Withdrawal of antacid and anticholinergic drugs
(12 h), and of PPIs (1 week) is recommended [222]. The secretin test is positive when
a >120 pg/mL increase of FSG over the basal value is found (sensitivity 94 %, specificity
100 %) [88, 223]. Calcium stimulation test (5 mg/kg body weight per hour, infused
over 3 h, increase >395 pg/mL over the basal FSG as cut-off) may alternatively be
used. However, it is hampered by lower sensitivity, specificity and higher side effects
[88]. Gastric acid secretion stimuli are no longer performed [203].
Imaging: After biochemical diagnosis, EGDS is required. In ZES, peptic ulcer disease
is found distally to the duodenal bulb within the descending part of the duodenum
or even further distally within the jejunum. Peptic ulcers frequently occur in groups
indicating some substantial acid hypersecretion [84].
The following imaging procedures may be used to localize the primary tumor, determine
the extent of the disease, evaluate indication to surgery, and assess response to
treatments [88]: (1) contrast-enhanced CT and/or MRI, EUS; (2) functional imaging
(SRS, PET); and (3) selective intra-arterial calcium injection angiography [84, 88].
Accurate localization of the tumor can result in complete surgical resection, decreased
rate of developing lymph node metastases, and increasing survival [88, 222, 224–226]
(Fig. 10).
Fig. 10
Diagnostic flow-chart for suspected gastrinoma
Spontaneous hypoglycemia
Clinical approach: when to suspect a GEP-NEN
Hypoglycemia (plasma glucose <60 mg/dL on a venous blood sample) is an uncommon clinical
problem in non-diabetic adults. The presence of symptoms reinforces the clinical relevance
of this finding because some normal subjects may have an asymptomatic low glucose
level after prolonged fasting. Symptoms may be due to sympathoadrenal activation (“adrenergic
symptoms”, i.e., sweating, shakiness, tachycardia, anxiety, hunger) and/or neuroglycopenia
(weakness, dizziness, inappropriate behavior, altered concentration, confusion, blurred
vision and, in extreme cases, coma and death) [227–229]. Symptoms may present at a
variable glucose level (generally as low as <55–60 mg/dL) [227, 228, 230, 231].
Hypoglycemia may be due to several conditions beyond insulin-secreting tumors [232]
(Table 8).
Table 8
Differential diagnosis of hypoglycemia
Drugs
Insulin, oral hypoglycemic drugsQuinine, pentamidine, indomethacin, lithiumMore rarely:
ACE-inhibitors, levofloxacin, trimethoprim-sulfamethoxazole, and heparin
Excessive alcohol consumption
Block of stored glucose release
Liver, kidney or heart failure
Depletion of substrates required for gluconeogenesis
Long-term starvation (anorexia nervosa)
Depletion of substrates required for gluconeogenesis
Non-islet cell tumors
Excessive production of IGF-II that causes the use of too much glucose
Gastric surgery (post-gastric bypass)
Accelerated transit and malabsorption
Hypoadrenalism and hypopituitarism
Deficiency of hormones that regulate glucose production
Insulin autoimmune hypoglycemia
Insulinoma (Box 4) should be strongly suspected in presence of the Whipple triad,
which occurs in about 75 % of patients and combines (1) symptoms of hypoglycemia,
(2) low blood sugar concurrent with symptoms, and (3) reversal of symptoms after glucose
administration [233]. Neuroglycopenic symptoms usually dominate the clinical picture
so that insulinoma may be misdiagnosed with cognitive impairment, psychiatric illnesses
or seizure disorders. Frequently, the occurrence of bizarre behavior or confusion
states is more precisely described by concerned relatives or friends than by the patient
himself. Adrenergic and neuroglycopenic symptoms may coexist, especially in the early
phase of the disease. A detailed description of pure adrenergic symptoms, however,
is more specific of a “functional hypoglycemia”.
Hypoglycemic symptoms occur most frequently at night and/or early morning and, anyway,
in a protracted fasting state. Yet, the occurrence of post-prandial hypoglycemia does
not exclude an insulinoma [234, 235]. Symptoms can be worsened by exercise, alcohol,
hypocaloric diet, and by concomitant clinical conditions or use of drugs (see above)
[236, 237]. Weight gain occurs in 20–40 % of patients that may develop overweight
because of hyperinsulinism.
Box 4
Insulinoma
[
236
,
237
]
Insulinoma is a NEN arising from insulin-secreting cells in pancreatic islets. Other
hormones and metabolites (gastrin, ACTH, glucagon, hCG, somatostatin, and 5-HIAA)
may be also secreted from this neoplasm.
About 90 % of insulinomas are benign. In rare cases neither a single nor multiple
tumors can be identified and the syndrome depends on diffuse beta-cell hyperplasia.
In malignant forms with liver metastases, a 16–26 months survival is to be expected.
Only 5 % of all insulinomas are associated with MEN-1; in case of multiple insulinomas
(near 10%), MEN-1 prevalence raises to 50 %.
Work-up in the patient with suspected insulinoma
Biochemical assessment: Symptoms and/or signs suggesting hypoglycemia combined with
a ≤55 mg/dL (3.0 mmol/L) plasma glucose, a ≥3.0 μU/mL (18 pmol/L) plasma insulin,
a ≥0.6 ng/mL (0.2 nmol/L) C-peptide, and a ≥5.0 pmol/L proinsulin indicate endogenous
hyperinsulinism [227, 228, 231]. Exogenous insulin-induced hypoglycemia is always
associated with low levels of C-peptide. In patients with insulinoma, proinsulin corresponds
to about 70 % of insulin immunoreactivity, whereas it is normally limited to 20 %.
Blood and urine assays for sulfonylureas will detect factitious hypoglycemia caused
by these drugs. Pituitary and adrenal function tests are useful to rule out hypoadrenalism
and hypopituitarism [227, 228, 230, 238].
Provocative tests: Biochemical diagnosis is based on lack of suppression of endogenous
insulin secretion by hypoglycemia [239] and inappropriately elevated insulin level
during hypoglycemia is the diagnostic key point. In 95 % of cases, the diagnosis is
achieved only during prolonged fasting (up to 72 h) inducing symptomatic hypoglycemia
[240]. The test should be performed on inpatients under close supervision and with
regular control of glycaemia and mental status. A ≥3 µU/mL (≥18 pmol/L) insulin value,
in the presence of glucose level <55 mg/dL has recently been proposed as diagnostic
cut-off [223]. Plasma β-hydroxybutyrate levels ≤2.7 mmol/L may confirm the diagnosis,
demonstrating the suppressive effect of insulin on ketogenesis even during a protracted
fasting [231].
At the end of the 72-h fasting test, in the absence of hypoglycemia, the use of stimulation
tests was proposed [231]. Stimulation tests, e.g., tolbutamide, glucagon or calcium,
are not recommended because they may induce a prolonged and refractory hypoglycemic
condition, but long-term fasting can be finished after 72 h with bicycle test.
Imaging: In all patients with a confirmed biochemical diagnosis, imaging is indicated
to localize the tumor [241].
Since 80 % of insulinomas are <2 cm in size, they are frequently missed by high-resolution
transabdominal US (50 % sensitivity), while EUS is more sensitive (77 %) and should
be preferred [242]. Helical or multislice CT and MRI offer a comparable (82–94 %),
but incomplete, sensitivity [243, 244]. Selective arteriography has an 82 % sensitivity
and a 95 % specificity.
Due to small size and/or lack of SSTR2 expression in 50 % of insulinoma [151], SSTR-related
imaging plays a minor role than morphological imaging. DOPA-PET has been proposed
as an alternative tool [245]. Radiolabelling with 111In-labeled glucagon-like peptide-1
receptors agonist (111In-DOTA-exendin-4) is a promising technique, still not routinely
used [246].
Arteriography combined with selective calcium stimulation: Calcium is able to stimulate
insulin release from neoplastic tissue, but not from normal islets. Hence, the catheterization
of the arterial branches of the celiac system and the measurement of insulin in the
blood sampled from hepatic veins during selective intra-arterial calcium injection
localize the pancreatic area nesting the tumors in 88–100 % of cases [34, 247, 248].
This test is cumbersome, expensive and poorly available. Accordingly, it should be
reserved only to selected, biochemically proved cases with negative imaging studies.
In spite of all the above reported diagnostic techniques, only 60–70 % of patients
have a successful preoperative localization. In patients with less threatening symptoms
that are fairly controlled by medical treatment a close surveillance may be advisable.
In severely symptomatic cases, the use of intraoperative US and the pancreatic exploration
conducted by an experienced surgeon identifies more than 90 % of the insulin-secreting
tumors [242, 249] (Fig. 11).
Fig. 11
Diagnostic flow-chart for suspected insulinoma
Work-up in the patient with metastatic disease and unknown primary tumor
Unknown primary NEN (UPN) is a condition of metastatic histologic or cytological confirmed
NEN without evidence of a primary site after a first diagnostic work-up, including
chest-abdomen CT scan, SRS, and upper and lower endoscopy.
The frequency of well-differentiated UPNs ranges from 9 to 19 % [250, 251]. The presence
of liver metastases largely influences prognosis in all types of NENs and is dependent
on primary tumor site, tumor extent (T-stage), and histologic differentiation (NET
vs. NEC). Reported survival rate at 5 years of G1–G2 small intestinal and pancreatic
NENs in the SEER database is 54 and 27 %, respectively [252]. Furthermore, survival
is reportedly worse in UPN patients as compared to patients with liver metastases
whose primary NEN is known [253]. In liver metastatic patients survival rate is influenced
by the presence of obstructive symptoms or symptoms related to peptide secretion.
The evaluation of a patient with UPN should encompass a detailed clinical history,
including family history to identify affected relatives and a patient’s increased
risk for endocrine tumors (i.e., MEN type 1 or 2), laboratory and radiographic studies
[254].
Histologic preparations should be reevaluated by IHC to guide the search for the primary
tumor: TTF-1 (pulmonary or medullary thyroid carcinoma), CDX-2 (intestinal), PAX-8,
histidine-decarboxylase (pancreatic), xenin (duodenal), gastrin (occult gastrinoma),
and PP/glucagon (pancreatic) [38, 255]. Biochemical work-up may include 5-HIAA, gastrin,
and other locally available tumor markers [256].
It has been recently reported that most UPNs are derived from pancreas and small bowel
[257]. Accordingly, further investigations for localizing the primary site in well-differentiated
NENs might include abdomen MRI, EUS, enteroCT/MR, 68Ga-PET, VCE, DBE to be shared
within a multidisciplinary team according to clinics, local availability and expertise
[124, 258, 259]. In NECs 18F-FDG-PET may be useful (Fig. 12)
Fig. 12
Diagnostic flow-chart in the patient with metastatic disease and unknown primary tumor
When and how to stage a previously diagnosed GEP-NEN
Evaluation of disease extension has a pivotal role in treatment planning.
Pre-treatment staging should include morphologic and functional imaging. Morphological
imaging is required for all GEP-NENs, irrespectively of their grade. SSTR-based functional
imaging (SRS or 68Ga-DOTA-peptide-PET) should be used for low-/intermediate-grade
GEP-NENs (WHO 2010 G1-G2), whereas 18F-FDG-PET should be preferentially used in G3
GEP-NENs and in some G2 cases.
For morphologic staging, a chest-abdomen-pelvis multidetector CT or a chest basal
CT plus abdomen-pelvis MRI should be used [87]. For functional staging, SRS using
111In-pentetreotide (Octreoscan®) is presently regarded as the gold standard. However,
if available, 68Ga-DOTA-peptide-PET with simultaneous CT should be preferred to SRS.
In facts, PET lacks the anatomic details required for therapeutic stratification (surgical
planning or dose calculation for radioembolization with radiolabeled microspheres).
Recently, MRI with liver-specific contrast combined with 68Ga-DOTA-peptides-PET has
been reported to be more accurate than PET-CT to detect GEP-NEN hepatic metastases
[260].
18F-DOPA-PET-CT and 11C-5HTP-PET-CT are promising tools. Their use might be considered
if results of SRS or 68Ga-DOTA-peptides-PET are negative [261].
Gastric NENs In small (<1 cm) type 1 and type 2 tumors, EGDS is usually the only recommended
imaging procedure [153]. Tumor invasiveness through the gastric wall must be evaluated
with EUS study: it is recommended before resection for polyps >1 cm in diameter. EUS
is also useful in assessing regional lymph nodes involvement, and allows histological
confirmation by FNA. Type 1 tumors do not require either abdomen multislice CT or
MRI, or SRS/68Ga-DOTA-peptides-PET; these imaging studies should be performed for
type 2 and type 3 neoplasm staging.
Duodenal NENs EUS is useful before resection of polypoid lesions; multislice abdomen
CT or MRI should be performed to assess local and distant disease extension. In patients
with local advanced neoplasm and/or liver metastases, bone scan and MRI of spine and
pelvis should be performed [153].
Jejuno-ileum NENs Chest-abdomen-pelvis CT scan or chest basal CT scan and abdomen-pelvis
MRI, SRS or 68Ga-DOTA-peptides-PET should be performed looking for distant metastases
[73]. Liver CT scan should be performed by multislice and multiphase technique. Colonoscopy
should be performed to rule out synchronous colorectal carcinoma.
Colorectal NENs Chest-abdomen-pelvis multislice CT should be carried out. Endoanal/rectal
US is very useful for assessing preoperatively the depth of tumor invasion in the
rectal wall and regional lymph node involvement [173].
NF pNENs For morphologic staging a multislice/multiphase CT or fat-saturated T1-weighted
and delayed enhanced T1-weighted MRI can be performed and EUS with biopsy [262]. Afterwards,
SRS or 68Ga-DOTA-peptides-PET should be performed.
Conclusions
The management of patients with GEP-NENs poses a significant challenge to clinicians
from the very start of the diagnostic work-up. The wide heterogeneity of disease presentation,
with a majority of asymptomatic patients and poorly specific clinical pictures may
account for a delay in definite diagnosis and appropriate treatment. The present document
has, therefore, been drawn with the purpose of offering a practical guide to physicians
facing the suspicion of GEP-NENs, in light of the available clinical evidence and
experience. Of course, many questions are still to be fully answered and many others
still to be addressed in the near future, as we move forward to new promising techniques
and diagnostic tools. For these reasons, in spite of its goal as a state-of-the-art
update, our document has not been conceived as the repository of the “ultimate truth”
in the field of GEP-NENs diagnosis. Instead, much attention has been devoted to the
logical framework, which should back up the clinical reasoning. Furthermore, the diagnosis
of GEP-NENs is heavily based on the contribution of a wide range of know-how and skills
provided by different specialists. The core team may include a varying combination
of different specialists, according to the local expertise and facilities; nevertheless,
the pathologist plays a key role in the diagnosis and classification of GEP-NENs,
because his/her information is critical to guide the prognosis and treatment planning.
Hence, a multidisciplinary team model is recommended as the best opportunity to reach
an accurate, safe and cost-effective diagnosis, likely to improve the outcome of patients
with GEP-NENs.
In conclusion, the Italian Association of Clinical Endocrinologists (AME) hopes the
present Position Statement will constitute an effective tool in helping the clinical
management of patients with GEP-NENs. Further implementations and updates of this
document will follow as new evidence and progress in the field emerge.
Other members of AME oncologic endocrinology group Giorgio Borretta, Cuneo; Renato
Cozzi, Milan; Giuseppe Francia, Verona; Rinaldo Guglielmi, Albano Laziale; Gabriele
Luppi, Modena; Salvatore Monti, Rome; Silvia Nasoni, Albano Laziale; Micaela Pellegrino,
Cuneo; Anna Pia, Turin; Sara Pusceddu, Milan; Valeria Ramundo, Naples; Francesco Scavuzzo,
Naples; Alessandro Scoppola, Rome; Ettore Seregni, Milan; Francesca Spada, Milan;
Laura Tonutti, Udine; Vincenzo Toscano, Rome; Maria Chiara Zatelli, Ferrara.