Introduction
Pancreatic cystic lesions (PCLs) are divided into two main classes, cystic mucinous
neoplasms (CMNs) and non-mucinous cysts. CMNs consist of intraductal papillary mucinous
neoplasms (IPMNs) and mucinous cystic neoplasm (MCNs). Mucinous and non-mucinous classification
is extremely important since these cysts are premalignant neoplasms with potential
for progression to pancreatic cancer.
Cross-sectional imaging methods and particularly endoscopic ultrasonography (EUS)
have a significant role in diagnosis and differentiation of PCLs [1]
[2]. However, there are some limitations to imaging alone and imaging may not be helpful
in differentiating between various types of cysts. EUS fine-needle aspiration (EUS-FNA)
of cystic lesions allows for biochemical and cytological analysis of cyst fluid that
might be further helpful for differentiation [2]
[3]. Among all cyst fluid diagnostic parameters, pancreatic cyst fluid (PCF) carcinoembryonic
antigen (CEA) concentration alone has been suggested as the most accurate test for
the diagnosis of CMNs at a cut-off level of 192 ng/mL. However, the diagnostic sensitivity
of PCF CEA at a cut-off level of 192 ng/mL has been reported to be less than 60 %
in recent clinical series [4]
[5]
[6].
The presence of KRAS mutation in PCF in recent studies has been suggested as a highly specific test for
the classification of a cyst as mucinous [7]
[8]
[9]. However, the number of patients included in some studies was small, with a limited
number of studies using histology as the diagnostic ‘reference standard [8]
[9]
[10]
[11]. Therefore, based on current reports, it is not certain whether routine testing
for KRAS mutations in PCF is warranted. Our study primarily aimed to assess the value of KRAS mutational analysis along with CEA in PCF for accurate diagnosis of mucinous cysts.
The study also sought to assess the value of KRAS mutation analysis in the differentiation of malignant CMNs.
Patients and methods
The study is a retrospective analysis of prospectively collected EUS-FNA data from
two academic medical centers, Massachusetts General Hospital, (MGH) and Indiana University
(IU). Patients gave their consent for FNA of the cyst and for fluid analysis, and
a cyst registry was approved by the Institutional Review Boards (IRB) at both institutions.
Consecutive patients who underwent an EUS-FNA for PCLs between 2006 – 2014 and who
had sufficient fluid for KRAS mutation analysis were enrolled in the study. Exclusion criteria included patients
with a bleeding tendency (INR > 1.5, partial thromboplastin time > 50 sec or platelets
< 50,000 mm3), a solid mass, a history of pancreatic cancer, acute pancreatitis, a high clinical
suspicion of a pseudocyst or pancreatic abscess, pancreatic ductal adenocarcinoma
with cystic degeneration, cysts of extra-pancreatic origin and fluid provided by FNA
of the main pancreatic duct.
The study data were recorded in a prospectively maintained database and the patients
were divided into two separate groups for all analysis according to their management.
Patients who underwent a surgical resection were included in the surgical cohort,
and patients who were managed non-surgically included in the clinical cohort.
EUS-FNA
Patients who had a PCL ≥ 10 mm on cross-sectional imaging, and who were referred for
EUS-FNA made up the study population. The location, size, number and morphology (mural
nodule, wall thickness, septations, adjacent mass, ductal communication) of cysts
was recorded. EUS-FNA was attempted if the cyst diameter was ≥ 10 mm and if the results
could potentially impact patient management. The PCF was triaged for cytological examination
and biochemical (CEA and amylase) analysis first and then for molecular testing. KRAS mutation testing was performed selectively when the fluid volume was more than 0.5 mL.
To allow comparison of accuracy with KRAS analysis, cyst fluid CEA level was not used as a reference to differentiate mucinous
cysts in the clinical cohort. A cut-off CEA value of 192 ng/mL was considered positive
for only diagnostic calculations.
Pathology and cytology
Cysts were classified by histological examination as either mucinous (main-duct (MD),
branch-duct (BD) or combined (COM) [IPMN or MCN]) or non-mucinous cysts (serous, pseudocyst,
neuroendocrine and others). IPMNs were further subclassified according to the degree
of dysplasia using 2010 WHO criteria [12]; (i) IPMN with low- or intermediate-grade dysplasia, (ii) IPMN with high-grade dysplasia
(HGD), and (iii) IPMN with an associated invasive carcinoma. Malignant cysts were
defined as cysts with either HGD or invasive carcinoma.
Cytological analyses of PCF were reported as non-diagnostic (because of insufficient
cells or contamination) or diagnostic (adequate for evaluation of cellular elements).
Diagnostic samples were classified either as mucinous (thick, colloid-like extracellular
mucin without cells, mucinous epithelium with cytoplasmic mucin, low-grade mucinous
epithelium in a transduodenal FNA, high-grade atypia or adenocarcinoma) or non-mucinous
[13]. Nonspecific gastric-foveolar type epithelium without thick extracellular mucin
in the transgastric FNAs was defined as nondiagnostic.
Classification of cysts
The mucinous/non-mucinous cyst classification of cysts in the surgical cohort was based on surgical histology results. The cysts in the clinical cohort were classified as mucinous if they had one of the cytological criteria (thick, colloid-like
extra-cellular mucin without cells, mucinous epithelium with cytoplasmic mucin, low-grade
mucinous epithelium in a transduodenal FNA, high-grade atypia or adenocarcinoma) or
one of the EUS criteria [14] (mural nodule, wall thickness, septations, adjacent mass, ductal communication).
These criteria are summarized in [Table 1]. The cysts without any cytological or EUS criteria for a mucinous cyst were classified
as non-mucinous.
Table 1
Diagnostic criteria for mucinous cysts in non-surgical cohort.
|
Endosonographic criteria
|
-
Mural nodule
-
Wall thickness
-
Septations
-
Adjacent mass
-
Ductal communication
|
|
Cytological criteria
|
-
Thick, colloid-like extracellular mucin without cells
-
Mucinous epithelium with cytoplasmic mucin
-
Low-grade mucinous, epithelium in a transduodenal FNA
-
High-grade atypia or adenocarcinoma
|
Abbreviation: FNA, fine-need aspiration
KRAS analysis
KRAS 2-point mutation analysis on codons 12 and 13 was determined by fluorescent-based
direct sequencing (PathFinder TG®; Redpath, Pittsburgh, PA, USA) for all cases from IU, and for cases from MGH until
the year 2010 by using the method previously described [7]. Starting in 2010, KRAS analysis on codons 12 and 13 was performed by using a primer extension-based method
(SNaPshot® Life Technologies) in the Molecular Pathology Department of MGH. PCF with no amplification
or no mutation were accepted as KRAS mutation negative which represents wild-type KRAS.
Surgery
All patients with a clinical diagnosis of MCN, MD-IPMN, or COM-IPMN were referred
for surgical resection if they were good surgical candidates. Patients with BD-IPMNs
were evaluated and referred to surgery according to International Consensus Guidelines
from 2006 [15] and then 2012 [16].
Data analysis
The sensitivity, specificity, positive predictive value (PPV), negative predictive
value (NPV) and diagnostic accuracy of KRAS, CEA and their combination for the diagnosis of a mucinous cyst were calculated.
The true positivity and false positivity was accepted with the positivity of one of
the tests. Receiver-operator curves were generated and the area under the curve (AUC)
calculated for diagnostic value of the tests. AUC values of tests and their combinations
were compared statistically. Comparisons between categorical variables were tested
with Chi Square test. Independent Student t-test was used to compare mean values between two independent groups. The agreement
between CEA and KRAS to differentiate mucinous cysts was measured by concordance correlation coefficient.
An rc value of 1 represents perfect agreement and 0 represents the agreement expected from
chance. A 2-tailed P value < 0.05 was considered statistically significant. SPSS 15.0 package program
(2006 SPSS Inc., Chicago, IL) was used for statistical analysis.
Results
A total of 2750 patients underwent an EUS-FNA of a pancreatic cyst during the study
period, of whom data on 943 with a KRAS mutation analysis result were available for analysis (the molecular analysis results
of 282 cases from IU have been previously reported [17]). Patients were excluded from KRAS testing due to insufficient PCF volume, the gastroenterologist’s decision not to
test for KRAS mutation which was based on clinical findings, or due to technical failures of the
test.
One hundred and forty-seven patients with a KRAS mutation analysis underwent surgical resection and made up the surgical cohort. The
remaining 796 cases were classified by EUS findings or cytological results and they
made up the clinical cohort. A flowchart of the study population is shown in [Fig. 1]. Mucinous cysts constituted 73.4 % (108/147) of patients in the surgical cohort
and 67.7 % (539/796) of patients in the clinical cohort. Cyst fluid CEA level > 192 mg/dL
plays a key diagnostic role in mucinous cyst definition but we did not use it for
the basic classification to avoid a flaw in the methods. However, the addition of
CEA criteria to cytology plus EUS for a mucinous definition added 11 more cases to
the mucinous group in the clinical cohort. These changes did not affect the overall
performance of KRAS or KRAS + CEA in this cohort.
Fig. 1 Flowchart of the study population.
Baseline demographics, clinical and cyst characteristics of the study cohorts, based
on mucinous classification, are provided in [Table 2].
Table 2
Demographic, clinical and cyst characteristics of the study cohorts based on the mucinous
classification.
|
|
Surgical cohort
|
Clinical cohort
|
|
Total
|
Mucinous
|
Non-mucinous
|
p
|
Mucinous
|
Non-mucinous
|
p
|
|
Number (n)
|
943
|
108
|
39
|
|
539
|
257
|
|
|
Mean age (range), years
|
66.4 (17 – 95)
|
61.9 (20 – 89)
|
56.4 (27 – 77)
|
P = 0.053
|
70.3 (17 – 95)
|
61.2 (23 – 88)
|
P = 0.0001
|
|
Gender, male/female
|
384/559
|
36/72
|
17/22
|
P = 0.68
|
222/317
|
109/148
|
P = 0.8
|
|
Symptomatic
[1]
, n (%)
|
524 (56.2)
|
70 (64.8)
|
21 (53.8)
|
P = 0.42
|
296 (54.9)
|
143 (55.6)
|
P = 0.9
|
|
Amylase > 250 mg/dL, (%)
|
68.2
|
55.7
|
53.1
|
P = 0.97
|
74.5
|
63.7
|
P = 0.02
|
|
Cyst size (mean diameter ± SD), mm
|
25.3 ± 16
|
27 ± 15
|
23 ± 14
|
P = 0.45
|
24 ± 17
|
22 ± 15
|
P = 0.13
|
|
Cyst location (%)
Head/neck/Unc.
Body
Tail
|
55
24
21
|
40
24
36
|
32
21
47
|
P = 0.69
|
58
25
17
|
55
23
22
|
P = 0.48
|
|
Cyst number (%)
Single
Multiple
|
69
31
|
70
30
|
75
25
|
P = 0.95
|
66
34
|
74
26
|
P = 0.09
|
1 One of the symptoms referable to the pancreas; abdominal pain, weight loss, obstructive
jaundice, pancreatitis.
Surgical pathology and cytology
Mucinous cysts were malignant in 27 (invasive carcinoma in 19, and high-grade dysplasia
in 8) of the patients based on the surgical pathology. Malignancy was identified in
three cases of MCN. Cytological evaluation was non-diagnostic in 201 (25.2 %) of the
clinical cohort because of inadequate or acellular material. A mucinous cyst was defined
in 26 % (207/796) of the clinical cohort by cytology (adenocarcinoma in 15, high-grade
atypia in 14, and by other cytological criteria in 178 cases) and 41.7 % (332/796)
by EUS criteria. Two hundred and fifty-seven patients in the clinical cohort (32.3 %)
had none of the cytological or EUS criteria for a mucinous cyst, and their disease
was classified as non-mucinous.
KRAS and CEA diagnostic results
The sensitivity, specificity, PPV, NPV, diagnostic accuracy and AUC value of KRAS positivity, CEA elevation and their combination for the diagnosis of a mucinous cyst
as a whole and for each cohort are provided in [Table 3]. The PCF CEA level at a 192 ng/mL cut-off value was highly specific at 93.2 % but
the sensitivity was found to be 56.3 % in the whole group. The sensitivity of PCF
CEA for a mucinous cyst diagnosis was lower in the clinical cohort than in the surgical
cohort (54.6 % vs. 63.2 %, P = 0.03). The sensitivity of KRAS for a mucinous cyst diagnosis was 46.3 % (50 /108) and 48.8 % (263 /539) for the
surgical and clinical cohorts, respectively, and the specificity was 100 % for both.
Adding the KRAS result to the CEA result increased the sensitivity to 13.8 % in the surgical cohort
and 21 % in the clinical cohort. The diagnostic accuracy of the combined tests was
more than 80 % in all cohorts. The increased diagnostic accuracy and AUC with the
KRAS-CEA combination was statistically significant compared to KRAS and CEA alone in the clinical cohort. The study parameters and assay results based
on histological subtypes are shown in [Table 4]. The KRAS positivity rate was higher in patients with IPMN than in those with MCN but the results
were not statistically significant (49.4 % vs. 33.3 %, P = 0.27). KRAS mutation was positive in 12 patients in the surgical cohort and in 74 patients in
the clinical cohort when CEA level is under 192 mg/dL. The correlation coefficient
rc was 0.23 for the surgical and 0.28 for the clinical cohorts, which demonstrated
a weak correlation between KRAS and CEA results. The KRAS positivity and CEA levels did not show any correlation with the size, number and location
of the cysts.
Table 3
Diagnostic value of CEA and KRAS to differentiate mucinous vs. non-mucinous cysts.
|
All patients n = 943
|
Surgical cohort n = 147
|
Clinical cohort n = 796
|
|
Test
|
KRAS+
|
CEA↑
|
KRAS+ or CEA↑
|
KRAS+
|
CEA↑
|
KRAS+ or CEA↑
|
KRAS+
|
CEA↑
|
KRAS+ or CEA↑
|
|
Sensitivity %
|
48.3
|
56.3
|
75.8
|
46.3
|
63.2
|
77
|
48.8
|
54.6
|
75.6
|
|
Specificity %
|
100
|
93.2
|
93.2
|
100
|
90.9
|
91.1
|
100
|
93.8
|
93.8
|
|
PPV %
|
100
|
95.4
|
96.6
|
100
|
94.8
|
95.7
|
100
|
95.6
|
96.8
|
|
NPV %
|
47
|
45.9
|
60.6
|
40.2
|
48.3
|
60.7
|
48.2
|
45.2
|
60.5
|
|
Accuracy %
|
64.5
|
66.8
|
80.9*
|
60.5
|
70.8
|
81.6^
|
65.3
|
65.8
|
80.8[1]
|
|
AUC
|
0.742
|
0.748
|
0.846*
|
0.730
|
0.781
|
0.838~
|
0.744
|
0.742
|
0.847[1]
|
Abbreviation: PPV, positive predictive value; NPV, negative predictive value; AUC,
area under curve
1
P < 0.0001 vs. KRAS + and CEA↑ alone, ^P
< 0.01 vs KRAS + , > 0.05 vs CEA↑ alone, ~
P > 0.05 vs. KRAS + and CEA↑ alone
Table 4
Study parameters based on histological cyst type (n = 147).
|
Parameters
|
IPMN
|
MCN
|
Serous
|
Pseudocyst
|
NET
|
Others
|
|
Number (n)
|
87
|
21
|
9
|
8
|
12
|
10
|
|
Mean age (range), years
|
66.3 (29 – 89)
|
45.3 (20 – 70)
|
51.8 (27 – 75)
|
57.8 (30 – 66)
|
62.2 (40 – 77)
|
53.1 (28 – 70)
|
|
Gender, male/female
|
35/52
|
1/20
|
3/6
|
4/4
|
8/4
|
2/8
|
|
KRAS (+), %
|
49.4
|
33.3
|
0
|
0
|
0
|
0
|
|
CEA > 192 mg/dL, (%)
|
61.9
|
68.4
|
0
|
12.5
|
16.6
|
10
|
|
KRAS (+) or CEA ↑ (%)
|
77
|
73.6
|
0
|
12.5
|
16.6
|
10
|
|
Amylase > 250 mg/dL, (%)
|
53.7
|
64.7
|
37.5
|
75
|
25
|
77
|
Abbreviations: IPMN, intraductal papillary neoplasms; MCN, mucinous cystic neoplasm; NET, neuroendocrine
tumor
KRAS and CEA in malignant cysts
Based on histology and cytology, there were 56 patients with a malignant cyst (34
patients with adenocarcinoma and 22 patients with high-grade dysplasia/atypia) in
the study population. The surgical cohort had 19 patients with adenocarcinoma and
8 patients with high-grade dysplasia. The clinical cohort had 15 patients with adenocarcinoma
and 14 patients with high-grade atypia. KRAS mutation was positive in 73.2 % (41/56) of malignant cysts. However, KRAS mutations were found in 37.3 % (31/83) of surgically confirmed benign mucinous cysts.
KRAS mutations were significantly more frequent in malignant mucinous cysts compared to
benign mucinous cysts (73.2 % vs. 37.3 %, P = 0.001). Having a KRAS mutation in a mucinous cyst increased the relative risk of a malignant cyst to 1.96
(1.42 < RR < 2.70) and odds ratio to 5.58 (2.06 < OR < 10.32). In malignant cysts,
KRAS mutation rate was higher in patients with adenocarcinoma (28/34, 82.3 %) than patients
with high-grade dysplasia/atypia (13 /22, 59 %). The study parameters between malignant
cysts and surgically confirmed benign cysts are shown in [Table 5]. Elevated PCF CEA level was found more frequently in malignant mucinous cysts compared
to benign cysts (78.5 % vs. 57.8 %, P = 0.02). The median CEA values of malignant cysts were also significantly higher
than non-malignant cysts (921 mg/dL vs. 491 mg/dL, P = 0.04).
Table 5
Study parameters in patients with malignant vs. non-malignant mucinous cysts.
|
Parameters
|
Malignant mucinous cysts (surgical pathology or cytology)
|
Non-malignant mucinous cysts (surgical pathology)
|
P
|
|
Number (n)
|
56
|
83
|
|
|
Mean age (range), years
|
73.1 (44 – 91)
|
62.3 (20 – 89)
|
P = 0.002
|
|
Gender, male/female
|
20/36
|
24/59
|
P = 0.6
|
|
KRAS (+), %
|
73.2
|
37.3
|
P = 0.001
|
|
CEA (median, mg/dL)
|
921 (4.5 – 168.140)
|
491 (0.3 – 48.378)
|
P = 0.04
|
|
CEA > 192 mg/dl, (%)
|
78.5
|
57.8
|
P = 0.02
|
|
KRAS (+) or CEA ↑ (%)
|
91
|
69.8
|
P = 0.01
|
|
Amylase > 250 mg/dL, (%)
|
29.8
|
59.4
|
P = 0.01
|
The combination of KRAS and CEA revealed 91 % sensitivity but 30.1 % specificity for a malignant cyst diagnosis.
The NPV of KRAS mutation alone and together with CEA elevation was 77.6 % and 83.3 %, respectively,
in differentiating non-malignant from malignant CMNs.
Discussion
The accurate diagnosis of PCLs is a key challenging issue for optimal clinical management.
The high specificity of KRAS mutation for mucinous cysts has been confirmed in subsequent clinical studies but
low sensitivity limited its widespread usage in routine PCF analysis [10]
[18].
To our knowledge, the current study includes the largest cohort of KRAS mutation testing for CMN diagnosis. The results of our study confirmed high specificity
of KRAS mutation for MCNs diagnosis in both surgical and clinical cohorts. However, the sensitivity
of KRAS mutation alone was limited. The PCF CEA (cutoff 192 ng/mL) was also highly specific
for mucinous cysts but the sensitivity was found to be lower than previously reported
by the Cooperative Study [14]. The sensitivity of CEA for mucinous cysts diagnosis in the surgical cohort was
higher than in the clinical cohort in our study. This might be a result of a selection
bias of patients for surgery including malignant mucinous cysts and MCNs. A meta-analysis
of 12 published studies showed that the pooled sensitivity and specificity of CEA
for the differentiation between mucinous and non-mucinous cystic lesions was 63 %
and 88 %, respectively [19]. Most of these studies included surgically resected lesions because there is no
optimal diagnostic standard for diagnosis of mucinous cyst in a clinical setting.
As a result, these published series usually consisted of patients with malignant IPMNs,
MCNs and BD-IPMNs with high-risk stigmata or worrisome features. In our study, we
have found that CEA elevation was significantly more common in malignant mucinous
cysts compared to non-malignant mucinous cysts.
The additive value of KRAS mutation testing to PCF CEA may have potential to increase the sensitivity for diagnosis
of mucinous cysts. The combination of elevated CEA and KRAS mutation detected an additional 11 mucinous cysts in 76 cases in the PANDA study
[7]. In another study, the sensitivity of KRAS mutation and elevated CEA for mucinous differentiation were 54 % and 62 %, respectively,
and the combination of both improved the sensitivity to 83 % and maintained a high
specificity of 85 % [10]. In our study, the addition of KRAS analysis to CEA increased the sensitivity and diagnostic accuracy of CEA significantly
without decreasing specificity. The ROC analysis also showed significantly larger
AUC values with the combination of tests. However, we could not demonstrate a statistical
difference in diagnostic parameters with use of combination tests in the surgical
cohort. In fact, sensitivity, diagnostic accuracy and AUC values of combined tests
were very similar between the surgical and the clinical cohorts. There could be two
reasons for the lack of a statistical significance in the surgical cohort. First,
the sensitivity of CEA alone for diagnosis of a mucinous cyst was higher in the surgical
cohort than in the clinical cohort, which may have limited the added benefit of KRAS analysis to the CEA testing. Second, the sample size was smaller in the surgical
cohort than in the clinical cohort, which decreased the power of the analysis to detect
a statistical significance.
To detect the malignant potential of pancreatic cysts accurately is the ultimate goal
of any diagnostic test. KRAS mutation was shown as one of the earliest genetic alterations in the majority of
pancreatic ductal adenocarcinomas but its role and presence in the development of
malignant cyst lacks clarity. The role of cyst fluid KRAS mutation for assessment of malignancy was first studied in 11 patients with malignant
cysts, and was reported as positive in 10 of these cases [20]. The number of DNA mutations was found to be higher in malignant cysts in the PANDA
study but the presence of KRAS mutation alone did not significantly differ between premalignant and malignant cysts
[7]. The high-amplitude KRAS mutation followed by allelic loss was highly specific for the presence of malignancy
(96 %) but the sensitivity remained low (37 %). The presence of PCF KRAS mutation was associated with progression and development of malignancy in long-term
follow-up of 63 patients in another study by the same group [21]. Our data showed a high frequency of KRAS mutation in malignant cysts. Unlike the PANDA study, both low- and high-clonality
mutations of KRAS were accepted as positive in our analysis. This may explain the higher sensitivity
and lower specificity of KRAS mutation in our study for malignant cyst detection. Another interesting finding in
our study was that cysts with high-grade dysplasia/atypia had a higher KRAS mutation rate than did non-malignant cysts but a lower rate than adenocarcinomas.
The number of cases with high-grade dysplasia/atypia was too small to make a conclusion,
but a possible role of KRAS mutation in adenocarcinoma sequencing seems likely. KRAS mutation may be one of the important tests predicting mucinous cyst behavior, but
our data show that the diagnostic value of KRAS positivity alone for malignant cysts remains limited since it lacks specificity for
malignant non-malignant differentiation. Nevertheless, high NPV might be helpful to
exclude a malignant cyst in clinical practice. The combination of KRAS positivity with CEA does not add diagnostic accuracy for malignant cysts but sensitivity
and NPV increased to 90.9 % and 80.9 %, respectively. The integration of molecular
pathology with cytology, imaging and fluid chemistry determined the diagnosis of malignancy
with a 90.6 % sensitivity, 83.3 % specificity and 89.6 % accuracy [22].
GNAS mutation has been detected in PCF samples of patients with IPMN but not in fluid
samples of other PCLs in recent studies [23]. It has high specificity but a limited sensitivity for a diagnosis of IPMN. The
combination of KRAS and GNAS mutation analyses improved the sensitivity of molecular testing in preliminary studies
[23]
[24]; however, more data are still needed to demonstrate the benefit of a combination.
There are certain limitations to this study. Because surgical pathology was the reference
standard for a mucinous cyst diagnosis, the results of the surgical cohort, no doubt,
will be more valuable. Our study included a significant number of patients but the
majority of them were in the clinical cohort in whom a final mucinous/non-mucinous
classification was solely based on EUS and/or cytological criteria. To avoid introducing
bias to the methods, we did not use CEA level and KRAS mutation for cyst classification in the clinical cohort. The specificity of cytology
for mucinous cyst diagnosis using the defined study criteria is very high; however,
it underestimates the number of mucinous cysts due to low sensitivity [3]. The EUS criteria used to define mucinous cysts are useful in clinical practice
but their specificity and sensitivity are also limited. Therefore some of the mucinous
cysts may have been included in the non-mucinous group.
On the other hand, the study was conducted in two tertiary referral high-volume centers
where all EUS were performed by highly skilled endosonographers in the field. In clinical
practice, only a small proportion of pancreas cysts patients undergo surgery, thus
limiting the sample size for an adequate power analysis. For this reason, well-designed
studies need to include patients with a non-surgical diagnosis. Besides, the results
of selected patients for surgery may be different than the unselected clinical cohort,
and surgical results may not be applicable to all patients with pancreatic cysts.
Therefore, we believe the sample size of our clinical cohort is sufficient to make
a conclusion about the utilization of KRAS analysis.
Another limitation of this study was that the KRAS testing was not studied in all cysts but on a selected basis. This may limit the
applicability of our results for all pancreatic cysts detected in EUS. The retrospective
analysis of the outcomes data from the study databases was another drawback of the
study. In spite of the prospective collection of data, some missing or insufficient
data could undermine the study objectives. The analysis of KRAS mutation by two different methods is another potential weakness of the study. However,
the comparison of the SNaPshot genotyping system with direct DNA sequencing showed
100 % consistency in the validation study [25]. Performance characteristics of different KRAS mutation analysis techniques including both methods were found to be highly concordant
[26]. We do not think the use of KRAS analysis based on two different methods has an impact on our study results because
both methods are reliable and were validated in different studies.
In summary, the detection of a KRAS mutation in PCF is a highly specific test for mucinous cysts. The addition of KRAS testing to CEA outperforms its sensitivity but does not improve the diagnostic accuracy
in the surgical cohort. KRAS mutations are more frequently detected in malignant mucinous cysts than in non-malignant
cysts but the diagnostic accuracy is limited because of low specificity. In conclusion,
our data do not support routine use of KRAS testing in traditional PCF analysis, but it might be potentially useful in selected
cases.
