Introduction
Pancreatic neuroendocrine tumors (PanNETs), despite having been steadily increasing
in both incidence and prevalence during the past few decades, are rare (1 per 100,000
population), representing 1 % to 2 % of all pancreatic neoplasms [1]
[2]. Preoperative diagnosis is important since a solitary small tumor without evidence
of metastatic spread may be suitable for pancreatic preserving surgery, such as enucleation,
rather than more extensive resection [3]
[4]. However, preoperative localization can be difficult, as these tumors are frequently
smaller than 2 cm in diameter. Consequently, conventional imaging methods such as
transabdominal ultrasound (US), computed tomography (CT), and magnetic resonance imaging
(MRI) may fail to accurately localise the tumor in 10 % to 40 % of patients [2]
[5]
[6]. Similarly, somatostatin receptor scintigraphy has a limited sensitivity of 62 %
to 86 % as well as false-positive rate of up to 12 % [6].
Endoscopic ultrasound (EUS) has been reported to be highly accurate for preoperative
localization of PanNETs, mainly primary insulinomas, which are frequently negative
on SRS, and was established as an alternative to more invasive methods such as angiography.
PanNETs are identifiable by EUS in 79 % to 95 % of suspected cases [7]
[8]
[9], and appear hypoechoic, round and homogenous, though they may occasionally be isoechoic
or hyperechoic with irregular margins ([Fig. 1]) [10]. EUS-guided fine-needle aspiration (EUS-FNA) can confirm the diagnosis cytologically
and provide information to guide the type of surgical intervention, which is particularly
useful in diagnosing non-functioning tumors [11]. However, the literature states that the sensitivity of EUS-FNA in the diagnosis
of PanNETs ranges widely, from 47 % to 95 % [12]
[13]
[14].
Fig. 1 a EUS image of hypoechoic well-defined pancreatic NET and b histological pattern of a well-differentiated pancreatic neuroendocrine tumor.
To overcome the limitations of EUS-FNA, fine-needle biopsy (FNB) has been developed
for use with EUS scopes, to obtain core biopsies and improve histological diagnoses.
The 19 G Quick-Core (Cook UK, Limerick) needle (TNB) was first released, but despite
its initial favorable results [15], more recent studies indicated that the overall efficacy profile of EUS-guided Quick-Core
needle biopsy appeared modest, with a reported diagnostic accuracy for pancreatic
lesions of 33 % to 61 % [16]
[17]. On the other hand, in a prospective study from our unit of 159 patients [18], the combination of both EUS-FNA and Quick-Core sampling modalities resulted in
higher diagnostic accuracy rates than either of the techniques alone, with reported
diagnostic accuracies of pancreatic FNA, TNB and FNA/TNB of 77 %, 56 %, and 83 %,
respectively. More recent FNB needles have been developed, such as the Procore (Cook
UK, Limerick, Ireland) and the Sharkcore (Medtronic plc, Minneapolis, Minnesota, United
States) needles, with encouraging results demonstrating the efficacy of EUS-guided
core sampling [19]
[20]
[21].
We herein report and compare the results of different EUS-guided sampling methods
on patients in which the final diagnosis was that of a pancreatic neuroendocrine tumor.
Patients and methods
Patients and procedures
A retrospective review of a prospectively maintained database collected during a 13-year
period (May 2004 – February 2017), at two tertiary hepato-pancreatico-biliary (HPB)
endoscopy units that share the same endosonographers, the Royal Free Hospital and
the University College London Hospital in London, was carried out. All clinical, endoscopic,
and pathologic details of patients who underwent EUS-guided cell/tissue sampling of
suspicious pancreatic lesions identified by pancreatic protocol CT or MRI were entered
into the electronic database. All results, including technical aspects of the EUS
procedures, were recorded.
EUS was performed under midazolam and fentanyl sedation, usually as a day-case procedure.
All procedures were carried out by well-experienced endosonographers (> 1000 procedures).
Lesions underwent EUS-FNA or EUS-FNB sampling, or a combination of the two, employing
different types of needles. Needle type and size, and number of passes, were decided
at the discretion of the endoscopist.
All EUS-FNAs were performed using a disposable 22G or 25G FNA needle (EchoTip, Cook
UK or Expect, Boston Scientific) in conjunction with linear-array echoendoscopes with
a 2.8 – 3.7mm accessory channel (Olympus GF-UC240P-AL5 and GF-UCT240-AL5, Keymed UK
Ltd, Southend on Sea, UK). After visualization of the target lesion endosonographically,
cell sampling was performed through the gastric or duodenal wall under real-time EUS
guidance. The needle was visualized throughout the procedure. During EUS-FNA, the
needle was moved back and forth during each pass, while applying suction using a 10-mL
syringe. Aspirated material was smeared onto glass slides and then alcohol fixed or
air-dried, while possible aspirated fluid or material was placed into a specimen container.
EUS-FNB (Quick-Core, Cook UK, Procore, Cook UK or Sharkcore, Medtronic) was initially
performed using the Quick-Core needles (Cook, UK) applying the same technique as reported
previously [15]
[16]
[17]. Briefly, the needles used had a 22 G or a 19 G outer cutting needle. Prior to use,
the Quick-Core needle was primed by pulling back the spring-loaded handle assembly
until it clicked into the “firing” position. This action drew the outer cutting needle
back, allowing the inner needle with the tissue tray to be advanced into the target
lesion under real-time EUS guidance, followed by firing of the spring-loaded cutting
needle over the tray. The needle was then withdrawn back into the sheath and the entire
assembly removed from the endoscope. Starting in 2011, both centers were equipped
with newly released FNB needles, such as the Procore (Cook UK) and Sharkcore (Medtronic)
needles. Using the more recent FNB needles, first the lesions were visualized endosonographically
and targeted with the needle. The needle was then moved back and forth during each
pass, while applying suction with a 10-mL syringe. After sampling, the biopsied tissue
from the needles was placed into a specimen container containing formalin. Adequacy
of sampling by EUS-FNB was defined by successful acquisition of at least one core
of tissue. There was no on-site microscopic evaluation of adequacy of EUS FNA or FNB
material by a pathologist.
Cytological and histological methods
The pancreatic specimens examined included cytological material obtained from the
EUS-FNA direct smears and liquid-based preparations and histological material obtained
from the EUS-FNA, formalin-fixed paraffin-embedded, cell-block, and core biopsy from
EUS-FNB. When only cytological material was available, diagnosis of PanNET was based
on compatible morphologic findings of characteristic cytologic and nuclear features
of the tumor cells, to include bland-appearing and monotonous cell morphology, round
to oval nuclei and classical salt-and-pepper chromatin distribution. However, cytological
diagnosis was not conclusive in such cases as no confirmatory immunohistochemistry
was possible and had to be integrated with clinical and radiological findings. Histologic
diagnosis of PanNET was definitive when cell-block obtained from EUS-FNA and core
biopsy obtained from EUS-FNB allowed immunohistochemical analysis demonstrating tumor
cell positivity for cytokeratins and neuroendocrine markers (chromogranin and synaptophysin).
Definitive diagnosis of PanNET was also provided when histological examination of
surgical specimens was available.
Negative FNA/FNB results without any evidence of malignancy were confirmed as NETs
during follow-up of at least 12 months by a multidisciplinary team specialized in
neuroendocrine tumors (currently part of the ENET network), mainly based on a combination
of clinical and imaging findings.
Outcomes assessment
The primary endpoint of the current study was diagnostic yield of various EUS-guided
tissue sampling, defined as the proportion of cases in which an adequate specimen
was obtained to pose a diagnosis, and assessment of variables possibly influencing
diagnostic yield. Secondary outcomes included complication rates during the procedures
and diagnostic sensitivity of various EUS-guided tissue sampling techniques, defined
as the proportion of cases in which diagnosis of PanNET was posed among the adequate
samples.
Statistical analysis
Continuous variables were described as median and interquartile range (IQR), whereas
ordinal and categorical variables were reported as proportions. Comparisons between
groups were performed with the Mann-Whitney U test and Kruskal-Wallis test for continuous
and ordinal variables. The Fisher’s exact test and its extension, also known as Freeman-Halton
test were used to compare categorical variables with two or more categories, respectively
[22]. Diagnostic yield and diagnostic sensitivity were computed with 95 %CI, and considered
significantly different between groups if 95 %CI did not overlap. In this case, statistical
significance was tested with Fisher’s exact test for confirmation. Statistical analyses
were performed with R version 3.4.2 for Mac [23].
Results
Of all patients who underwent EUS-FNA and/or EUS-FNB, 91 (M/F: 42/49) were diagnosed
with PanNET after a total of 102 procedures, which was confirmed by multidisciplinary
review and clinical follow-up. In 33 patients the diagnosis also was ascertained by
additional histopathological examination of surgical resection specimens. Analysis
of the technical aspects of pancreatic sampling during the 102 procedures revealed
that both EUS-guided sampling modalities were used in 28 procedures, while EUS-FNA
and EUS-FNB alone were used in 61 and 13 cases, respectively. Patient and lesion characteristics
are summarized in [Table 1].
Table 1
Patient, lesion and sampling characteristics.
|
Characteristics
|
|
|
42/49
|
|
|
57 (26 – 87)
|
|
|
18 (7 – 59)
|
|
Lesion diameter, group
|
|
|
27 (26.5 %)
|
|
|
33 (32.3 %)
|
|
|
42 (41.2 %)
|
|
Lesion location, n (%)
|
|
|
34 (33.3 %)
|
|
|
47 (46.1 %)
|
|
|
21 (20.6 %)
|
|
Sampling technique
|
|
|
61 (59.8 %)
|
|
|
13 (12.7 %)
|
|
|
28 (27.5 %)
|
FNA, fine-needle aspiration; FNB, fine-needle biopsy
Target lesions were localized in the uncinate process/head of the pancreas in 34 cases,
in the body in 47, while 21 were found in the tail. Median size of the lesions was
18 mm (range 7 – 59mm). EUS-FNA was performed using a 22G needle in the majority of
cases 74/89 (83 %), while a 25G needle was used in the remaining 17 %. Median number
of FNA needle passes was 3 (IQR 3 – 4). To perform EUS-FNB, 23 (56 %) 19G Quick-Core,
14 (34 %) Procore (12 22G and 2 25G) and 4 (10 %) 22G Sharkcore needles were used.
The median number of passes was three (IQR 3 – 4) during the EUS-FNB procedures.
The diagnostic yield (i. e. obtaining an adequate sample) of EUS-FNA alone and EUS-FNB
alone for the cytological/histological diagnosis was 77.5 % (95 %CI, 68.9 – 86.2 %)
and 85.4 % (95 %CI, 74.6 – 96.2 %), respectively. When the combination of both modalities
was used, adequate sampling was achieved in 27 of 28 cases, with a diagnostic yield
of 96.4 % (95 %CI, 89.6 – 100 %), being significantly higher than EUS-FNA alone (P = 0.023). In particular, among the 28 cases, seven FNA specimens were insufficient
for diagnosis, however, FNB samples were diagnostic for PanNET in six of them, while
in one case neither of the two sampling modalities was diagnostic. Non-significant
differences were observed when comparing performance of EUS-FNB needles: Quick-Core’s
diagnostic yield was 82.6 % (95 %CI, 62.9 – 93 %), whereas the more recent Procore
and Sharkcore needles had diagnostic yields of 85.7 % (95 %CI, 60.1 – 96 %) and 100 %
(95 %CI, 51 – 100 %), respectively (P = 0.99 for all of the comparisons).
No significant differences were observed for size and location of lesions, size of
needles, or number of passes. [Table 2] and [Table 3] report the variables possibly influencing diagnostic yield of PanNET for FNA and
FNB sampling.
Table 2
Exploratory analysis on variables possibly influencing EUS-FNA diagnostic yield.
|
Lesion and sampling characteristics
|
Adequate sample on FNA (n = 69)
|
Inadequate sample on FNA (n = 20)
|
P value
|
|
Lesion site, n (%)
|
0.789[1]
|
|
|
22 (31.9 %)
|
8 (40.0 %)
|
|
|
35 (50.7 %)
|
9 (45.0 %)
|
|
|
12 (17.4 %)
|
3 (15.0 %)
|
|
Lesion diameter, group
|
0.302[2]
|
|
|
22 (31.9 %)
|
3 (15.0 %)
|
|
|
20 (29.0 %)
|
8 (40.0 %)
|
|
|
27 (39.1 %)
|
9 (45.0 %)
|
|
|
3 (3 – 4)
|
3 (2 – 4)
|
0.101[3]
|
|
Size of needle, n (%)
|
0.094[4]
|
|
|
60 (87.0 %)
|
14 (70.0 %)
|
|
|
9 (13.0 %)
|
6 (30.0 %)
|
EUS-FNA, endoscopic ultrasound-guided fine-needle aspiration; IQR, interquartile range
1 Freeman-Halton test
2 Kruskal-Wallis test
3 Mann-Whitney U test
4 Fisher’s exact test
Table 3
Exploratory analysis of variables possibly influencing EUS-FNB diagnostic yield.
|
Lesion and sampling characteristics
|
Adequate sample on FNB (n = 35)
|
Inadequate sample on FNB (n = 6)
|
P value
|
|
Lesion site, n (%)
|
0.163[1]
|
|
|
11 (31.4 %)
|
4 (66.7 %)
|
|
|
14 (40.0 %)
|
2 (33.3 %)
|
|
|
10 (28.6 %)
|
0 (0 %)
|
|
Lesion diameter, group
|
0.439[2]
|
|
|
4 (11.4 %)
|
2 (33.3 %)
|
|
|
10 (28.6 %)
|
1 (16.7 %)
|
|
|
21 (60.0 %)
|
3 (50.0 %)
|
|
|
3 (2 – 4)
|
3 (2 – 4)
|
0.566[3]
|
|
Type of needle, n (%)
|
0.999[1]
|
|
|
19 (57.1 %)
|
4 (50 %)
|
|
|
14 (40.0 %)
|
2 (33.3 %)
|
|
|
2 (2.9 %)
|
0 (16.7 %)
|
|
Size of needle, n (%)
|
0.999[1]
|
|
|
19 (54.3 %)
|
4 (66.7 %)
|
|
|
12 (34.3 %)
|
2 (33.3 %)
|
|
|
4 (11.4 %)
|
0 (0 %)
|
EUS-FNB, endoscopic ultrasound-guided fine-needle biopsy; IQR, interquartile range
1 Freeman-Halton test
2 Kruskal-Wallis test
3 Mann-Whitney U test
Among the adequate samples, EUS-FNB specimens allowed a correct diagnosis of PanNET
in 33 of 35 cases (94.3 %, 95 %CI, 86.6 – 100 %). In one case autoimmune pancreatitis
was diagnosed and in another, no neuroendocrine tumor cells were seen. On the other
hand, FNA samples were diagnostic in 61 of 69 cases (88.4 %; 95 %CI, 80.9 – 96.0 %),
in five cases no neuroendocrine tumor cells were found, two cases were suggestive
for islet cell tumor and in one case pancreatitis was diagnosed. The combination of
both sampling modalities allowed a correct diagnosis of PanNET in 27 of 27 cases (100 %;
95 %CI, 100 – 100%).
Overall sensitivity of posing a correct diagnosis of PanNET based on the complete
set of samples of EUS-FNA alone and EUS-FNB alone was 68.5 % (95 %CI, 58.9 – 78.2 %)
and 80.5 % (95 %CI, 68.4 – 92.6 %), respectively. The combination of both modalities
achieved 96.4 % (95 %CI, 89.6 – 100 %) overall sensitivity, significantly higher than
EUS-FNA alone (P = 0.0021). There was only one reported complication where a patient developed a peripancreatic
hematoma after EUS-FNA associated with < 2 g/dL hemoglobin drop, and was admitted
to the hospital for observation and discharged 2 days later without requiring blood
transfusion or any further treatment.
Discussion
Preoperative diagnosis of PanNETs is important because it may alter clinical management
[24], based on the unique biological nature of PanNETs and the wide range of therapeutic
options available [25]
[26]. For example, in non-functioning PanNETs, patients often present with small pancreatic
lesions on cross-sectional imaging and preoperative histological confirmation could
lead to pancreas-preserving surgery. EUS has shown to be superior to CT for detecting
PanNETs, especially those 20mm. In a study from Khashab et al. [14], EUS detected 91 % of CT-negative tumours, with a mean size of 18.8 mm. Sensitivity
of EUS was significantly greater than that for CT (91.7 % vs 63.3 %) particularly
for insulinomas.
EUS-FNA can provide cytological diagnosis of pancreatic lesions smaller than 10 mm.
However, the accuracy of the technique is largely dependent upon the size and location
of the mass and expertise of the endoscopist [27]. EUS-FNA can also significantly increase the diagnostic impact in cases where PanNET
were not identified after initial cross-sectional imaging [28]. Immediate review of the aspirated material by an experienced pathologist can improve
adequacy of sampling as well as accuracy of cytological diagnosis [29], but this resource is not available in most institutions.
To date, there have been very few studies of EUS-FNA and of EUS-FNB in preoperative
diagnosis of PanNETs. In two studies of 10 and 30 patients [13]
[14], the accuracy of EUS-FNA was 90 % and 83 %, respectively. In another two larger
studies of 86 and 81 patients who had been diagnosed with PanNETs, 90 % had the diagnosis
established by EUS-FNA and the sensitivity was irrespective of tumor size or location
[30]
[31]. In both studies, EUS-FNB was not performed. Pais et al. [32] reported a similar sensitivity for EUS-FNA in diagnosis of functional and non-functional
PanNETs (77 % and 91 % respectively, P = 0.13), but diminished sensitivity (66 %) for tumors ≤ 15 mm. In an evaluation of
solid pancreatic masses [33], accuracy of EUS-FNA in patients with PanNETs (n = 15) was significantly lower than
that for adenocarcinomas (n = 59) (47 % vs 81 %, P < 0.01). In contrast, in the current study, the diagnostic yield of EUS-FNA in the
89 PanNET-positive patients was 77.5 %, similar to our previously published data in
patients with pancreatic adenocarcinoma [18]
[34].
There are several possible explanations for the lower accuracy of EUS-FNA in PanNETs
reported by other investigators. PanNETs are generally less than 2 cm in size [35], and it may be technically more difficult to obtain specimens from small lesions.
Moreover, cytological features of PanNETs, unlike the much more common adenocarcinomas,
are bland, resembling normal pancreatic acini. The most helpful morphological features
that enable cytological diagnosis of PanNET are a richly cellular aspirate with a
monotonous, poorly cohesive population of small cells with a speckled or dusty chromatin
pattern and plasmacytoid morphology. The final diagnosis rests largely on confirmation
of neuroendocrine differentiation of these cells by immunocytochemistry and can be
very accurate [36]
[37]. However, because PanNETs tend to be highly vascular, a bloody aspirate may dilute
cellularity and relatively poorly cellular samples prevent application of further
stains to confirm the diagnosis or grading of PanNETs; in a subset of patients in
the current study, grading was available for 16 of 32 FNA cases (50 %) but 13 of 14
FNB cases (93 %).
To overcome these limitations, the EUS-FNB technique was proposed as a method that
might replace or complement EUS-FNA [15]
[19]
[20]
[21]. EUS-FNB devices have been designed to improve tissue yield and to potentially achieve
a core histologic tissue specimen through EUS. However, lesions in the pancreatic
head and uncinate process are particularly difficult to biopsy [16]
[17]
[18], in part because of their anatomical position and fibrotic nature, as well as due
to needle rigidity, making at times trans-duodenal FNB puncture technically difficult.
In PanNETs the tissue is not as fibrotic as in adenocarcinomas and especially for
trans-gastric punctures, FNB is feasible. In the current series, six patients had
their diagnosis established only by FNB because EUS-FNA did not establish a diagnosis
because of inadequate (n = 5) or false-negative (n = 1) samples. Conversely, there
were six patients with false-negative (n = 2) and inadequate (n = 4) results with
EUS-FNB (all cases performed with Quick-Core needles), whereas FNA established the
diagnosis. Indeed, one limitation of EUS-FNB Quick-Core needle is the stiff firing
mechanism and 2-cm tray of the needle, making it difficult to obtain tissue cores
from small lesions in the head and uncinate process of the pancreas.
The more recent EUS-FNB devices have shown a higher diagnostic yield; smaller-diameter
and more flexible biopsy FNB needles are now available and may improve tissue acquisition,
particularly with a transduodenal approach [38]. In the study by Witt et al. the Sharkcore needles achieved a 90 % diagnostic sensitivity
with significantly fewer mean number of needle passes compared to EUS-FNA sampling,
although the study included only 20 patients [20]. Our results confirm the high diagnostic yield of the EUS-FNB devices (85.4 %) for
PanNET sampling, and also confirm that the combination of both FNA and FNB modalities
may be helpful in selected patients, such as cases suspicious for PanNETs, to achieve
a correct diagnoses [39].
The main limitation of this study is its retrospective design. Nevertheless, to date,
the literature comparing diagnostic efficacy of different FNA and FNB needles in diagnosis
of PanNETs has been limited. Most studies either include small study samples or compare
the needles for heterogeneous cohorts of pancreatic lesions. The current study includes
a reasonably large sample size, with more than 100 procedures, and focuses exclusively
on PanNET cases.
Conclusion
In conclusion, EUS-FNA and EUS-FNB are useful and safe methods for tissue diagnosis
in suspected PanNET. The addition of EUS-FNB needle biopsy improves the diagnostic
yield from 77.5 % for EUS-FNA alone to 96.4 % for EUS-FNA + FNB, and thus provides
additional clinical benefit to cytological assessment for preoperative diagnosis of
PanNETs.
