Endoscopic Ultrasound-guided Fine-needle Aspiration Samples Research Articles

Differential diagnosis between pancreatic neuroendocrine and solid pseudopapillary neoplasms on endoscopic ultrasound-guided fine-needle aspiration. An immunohistochemical study.

Objectives:To evaluate the role of applying a limited panel of immunohistochemical stains on the cellblock preparation from samples obtained by endoscopic ultrasound-guided fine needle aspiration (EUS-FNA) in the aim of differentiating solid pseudopapillary neoplasms (SPNs) from neuroendocrine tumors (NETs).Methods:We retrospectively retrieved all the EUS-FNAs of the pancreas that have a diagnosis of NET or SPN that were performed at 2 tertiary care hospitals in Riyadh, Kingdom of Saudi Arabia from May 2004 to December 2014. Diff-Quik, Papanicolaou, and Immunohistochemistry stains on cellblock preparations were performed.Results:Twenty cases were available (16 pancreatic neuroendocrine tumors (pNETs) and 4 SPNs). The pNETs were immunoreactive for synaptophysin, chromogranin A and CD56 while E-cadherin was diffusely to focally cytoplasmic positive. β-catenin was negative or showed focal cytoplasmic immunoreactivity. In comparison, SPNs were positive for vimentin, CD10, CD-56, focally positive for progesterone receptors and synaptophysin, and revealed nuclear immunostaining for β-catenin. They were negative for chromogranin A and E-cadherin.Conclusion:Based on EUS-FNA samples, nuclear immunoreactivity for β-catenin with loss of membranous immunostaining for E-Cadherin can potentially facilitate differentiating SPNs from pNETs.

MicroRNAs for Detection of Pancreatic Neoplasia: Biomarker Discovery by Next-generation Sequencing and Validation in 2 Independent Cohorts.

The aim of our study was to analyze the miRNome of pancreatic ductal adenocarcinoma (PDAC) and its preneoplastic lesion intraductal papillary mucinous neoplasm (IPMN), to find new microRNA (miRNA)-based biomarkers for early detection of pancreatic neoplasia. Effective early detection methods for PDAC are needed. miRNAs are good biomarker candidates. Pancreatic tissues (n = 165) were obtained from patients with PDAC, IPMN, or from control individuals (C), from Hospital Clínic of Barcelona. Biomarker discovery was done using next-generation sequencing in a discovery set of 18 surgical samples (11 PDAC, 4 IPMN, 3 C). MiRNA validation was carried out by quantitative reverse transcriptase PCR in 2 different set of samples. Set 1-52 surgical samples (24 PDAC, 7 IPMN, 6 chronic pancreatitis, 15 C), and set 2-95 endoscopic ultrasound-guided fine-needle aspirations (60 PDAC, 9 IPMN, 26 C). In all, 607 and 396 miRNAs were significantly deregulated in PDAC and IPMN versus C. Of them, 40 miRNAs commonly overexpressed in both PDAC and IPMN were selected for further validation. Among them, significant up-regulation of 31 and 30 miRNAs was confirmed by quantitative reverse transcriptase PCR in samples from set 1 and set 2, respectively. miRNome analysis shows that PDAC and IPMN have differential miRNA profiles with respect to C, with a large number of deregulated miRNAs shared by both neoplastic lesions. Indeed, we have identified and validated 30 miRNAs whose expression is significantly increased in PDAC and IPMN lesions. The feasibility of detecting these miRNAs in endoscopic ultrasound-guided fine-needle aspiration samples makes them good biomarker candidates for early detection of pancreatic cancer.

Diagnostic performance and factors influencing the accuracy of EUS-FNA of pancreatic neuroendocrine neoplasms.

BackgroundMultiple studies have investigated sampling adequacy of endoscopic ultrasound-guided fine needle aspiration (EUS-FNA) for pancreatic neuroendocrine neoplasms (pNENs). However, none have described the diagnostic performance of EUS-FNA for pNENs, or the influencing factors. The aim of this study was to evaluate the diagnostic accuracy of EUS-FNA, with post-operative pathological diagnosis as the gold standard, and factors predictive of inadequate EUS sampling.MethodsFrom 1998 to 2014, a total of 698 patients underwent pancreatic resection and 1455 patients underwent EUS-FNA sampling for pancreatic lesions. A total of 410 cases underwent both surgical resection and preceding EUS-FNA. Of these, 60 cases (49 true pNEN, nine non-diagnostic, two misdiagnoses) were included. We studied diagnostic performance of EUS-FNA and factors that were associated with failed diagnosis.ResultsOf the 60 cases, EUS-FNA yield was 49 true-positive cases, two misdiagnoses, and nine non-diagnostic cases (including six suggestive cases). Sensitivity, specificity, and accuracy were 84.5, 99.4, and 97.3 %, respectively; including the six suggestive cases, diagnostic values were 94.8 % sensitivity (55/58), 99.4 % specificity (350/352), and 98.7 % accuracy (405/410). In multivariate analysis, sampling adequacy rates were significantly lower when lesions were located in the pancreatic head [odds ratio (OR) = 10.0] and in tumor-rich stromal fibrosis (OR = 10.45). Tumor size, needle type, tumor grading, presence of cystic component, and time period were not significant factors.ConclusionsEUS-FNA offers high accuracy for pNEN. However, location of the tumor in the pancreatic head and presence of rich stromal fibrosis negatively impacts sampling adequacy.Electronic supplementary materialThe online version of this article (doi:10.1007/s00535-016-1164-6) contains supplementary material, which is available to authorized users.

Yield of endoscopic ultrasound-guided fine needle aspiration and endoscopic retrograde cholangiopancreatography for solid pancreatic neoplasms

Objective: Both endoscopic ultrasound-guided fine needle aspiration (EUS-FNA) and endoscopic retrograde cholangiopancreatography (ERCP) cytology may provide tissue diagnoses in solid pancreatic neoplasms. However, there are scant data comparing these two methods. This study aims at retrospectively comparing EUS-FNA and ERCP tissue sampling and ability of cytopathological diagnosis in solid pancreatic neoplasms and to determine usefulness and adverse events of combining both procedures. Material and methods: Two hundred and thirty four patients suspected to have solid pancreatic mass on abdominal ultrasound and/or computed tomography (CT) were enrolled. EUS-FNA (group A), ERCP cytology (group B) and combined procedures (Group C) performed in 105, 91 and 38 cases, respectively. Results: Sensitivity, specificity and accuracy were 98.9%, 93.3% and 98.1% for group A, and 72.1%, 60% and 71.4% for group B. Those for group C were all 100%. Sensitivity for malignancy in the pancreas head was 100% for group A and 82.4% for group B, and in the pancreas body and tail, 97.6% for group A and 57.1% for group B. EUS-FNA was more sensitive than ERCP cytology in diagnosing malignant pancreatic neoplasms 21–30 mm in size (p = 0.0068), 31–40 mm (p = 0.028) and ≥41 mm (p < 0.0001). Sensitivity for pancreatic malignancy with group C was 100% regardless of mass location or size. Adverse events were 1.9%, 6.6% and 2.6% following EUS-FNA, ERCP and combined procedures, respectively. Conclusions: EUS-FNA is superior to ERCP cytology for diagnosis of solid pancreatic neoplasms. Although combination of both procedures provide efficient tissue diagnosis and with a minimal adverse events rate, a prospective study including larger number of patients is required.

Efficacy of endoscopic ultrasonography and endoscopic ultrasonography-guided fine-needle aspiration for the diagnosis and grading of pancreatic neuroendocrine tumors

Background and aim: Pancreatic neuroendocrine tumors (pNETs) are histologically categorized according to the WHO 2010 classification by their mitotic index or Ki-67 index as G1, G2, or G3. The present study examined the efficacy of endoscopic ultrasonography (EUS) and EUS-guided fine-needle aspiration (EUS-FNA) in the diagnosis and grading of pNET. Methods: We retrospectively reviewed 61 pNETs in 51 patients who underwent EUS between January 2007 and June 2014. All lesions were pathologically diagnosed by surgical resection or EUS-FNA. We evaluated the detection rates of EUS for pNET and sensitivity of EUS-FNA, and compared the Ki-67 index between EUS-FNA samples and surgical specimens. EUS findings were compared between G1 and G2/G3 tumors. Results: EUS showed significantly higher sensitivity (96.7%) for identifying pNET than CT (85.2%), MRI (70.2%), and ultrasonography (75.5%). The sensitivity of EUS-FNA for the diagnosis of pNET was 89.2%. The concordance rate of WHO classification between EUS-FNA and surgical specimens was 69.2% (9/13). The concordance rate was relatively high (87.5%, 5/6) in tumors <20 mm but lower (57.1%; 4/7) in tumors ≥20 mm. Regarding EUS findings, G2/G3 tumors were more likely to be large (>20 mm), heterogeneous, and have main pancreatic duct (MPD) obstruction than G1 tumors. Multivariate analysis showed large diameter and MPD obstruction were significantly associated with G2/G3 tumors. Conclusions: EUS and EUS-FNA are highly sensitive and accurate diagnostic methods for pNET. Characteristic EUS findings such as large tumor size and MPD obstruction are suggestive of G2/G3 tumors and would be helpful for grading pNETs.

Suspicious cytologic diagnostic category in endoscopic ultrasound-guided FNA of the pancreas: Follow-up and outcomes.

The objective of the current study was to assess how the suspicious category is followed up in a large endoscopic ultrasound-guided fine-needle aspiration (EUS-FNA) service, its outcomes, and the predictors that are likely to be associated with the subsequent diagnosis of a neoplastic process. For pancreatic EUS-FNA samples with the suspicious category diagnosis, the authors reviewed the electronic medical record for the method of follow-up and the risks associated with pancreatic malignancy. Logistic regression analysis was used to determine the risk factors that were likely to be associated with the diagnosis of a neoplastic lesion after a cytologic diagnosis of "suspicious." Of a total of 3832 EUS-FNA cases, 116 were diagnosed with suspicious cytology. A total of 90 of 98 neoplasms (92%) were identified, including 72 carcinomas (73%). Similar rates of neoplasia were detected after repeat FNA (34 of 37 neoplasms [92%]) and subsequent biopsy/surgical resection (44 of 46 neoplasms [96%]), but significantly fewer neoplasms were detected among patients with clinical follow-up (18 of 23 neoplasms [78%]). On multivariate analysis of the potential predictive variables listed above, the presence of a mass was found to be significantly associated with a higher rate of diagnosis of a neoplasm, whereas weight loss was significantly associated with a diagnosis of carcinoma. The diagnostic category of "suspicious" is associated with a high risk of benign and malignant neoplasms, regardless of the method of follow-up. The presence of a mass and weight loss are significant predictors of a subsequent diagnosis of a neoplasm after suspicious cytology. In patients with suspicious cytology and these findings, surgery is recommended for resectable masses and repeat FNA for unresectable masses.

Abstract 1069: DNA methylation in circulating free DNA as a new biomarker for pancreatic cancer

Abstract Despite the clinical advancement, pancreatic cancer remains one of the most deadly cancers. Pancreatic cancers are often difficult to diagnose and there is no reliable screening biomarker for early detection of this cancer. Epigenetic alterations have emerged as a common hallmark of many types of human cancers as well as pancreatic cancers. Studies have shown that epigenetic dysregulation is associated with multiple steps during carcinogenesis. Therefore, detection of aberrant DNA methylation from blood samples may be a promising diagnostic tool for pancreatic cancers. First, we analyzed KRAS mutation status of 100 samples obtained by Endoscopic ultrasound-guided fine-needle aspiration (EUS-FNA), which is a safe and accurate technique to perform the pancreatic tissue biopsy, and found that more than 80% of the cells in the samples harbors KRAS mutation indicating high amount of cancer cells in the specimens. Next, we performed genome-wide DNA methylation analysis of 38 EUS-FNA specimens using Illumina Infinium HumanMethylation450 BeadChip. We further analyzed the deposited DNA methylation profiles of other tumor types in the public databases, such as TCGA, and identified 8 marker genes with high frequency and specificity of DNA methylation in pancreatic cancers. We validated the DNA methylation status of these genes by bisulfite pyrosequencing and quantitative methylation specific PCR. These markers were frequently methylated in EUS-FNA samples (95% of samples are methylation positive in at least one marker gene). Now we are analyzing the DNA methylation status in pancreatic cancers and corresponding blood samples in order to identify the most effective set of markers for diagnose pancreatic cancers. Our results suggest that analysis of DNA methylation in the circulating free DNA might be a safe and potent tool for evaluating and staging pancreatic cancers. Citation Format: Keiko Shinjo, Fumiharu Ohka, Keisuke Katsushima, Akira Hatanaka, Norihisa Ichimura, Zhao Juan, Kenji Yamao, Yutaka Kondo. DNA methylation in circulating free DNA as a new biomarker for pancreatic cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1069. doi:10.1158/1538-7445.AM2015-1069

Frequency and characterization of celiac ganglia diagnosed on fine-needle aspiration.

Introduction:Endoscopic ultrasound (EUS)-guided fine-needle aspiration (FNA) is frequently used to sample intra-abdominal lesions and lymph nodes. Celiac ganglia normally located near the celiac artery may be sampled during these procedures. The aim of this study was to determine the frequency of detection and cytologic findings of celiac ganglia diagnosed on FNA.Materials and Methods:A 14-year retrospective review of radiologic and endoscopic FNA cases involving the celiac region was performed. Cases in which ganglia were reported were further analyzed and slides reviewed.Results:A total of 354 patients underwent FNA of a suspected celiac lymph node (334 patients) or celiac mass (20 cases). In 9 of these patients (2.5%), ganglion cells were identified. These were identified in cases only after 2008 via EUS-guided FNA. Aspirates were hypocellular and bloody. Large ganglion cells were either sparsely dispersed or present in clusters. Ganglion cells had a low N: C ratio, granular cytoplasm with neuromelanin, and eccentric small round nucleus with a prominent nucleolus. One specimen had concomitant pancreatic adenocarcinoma. None of these cases had a false positive on-site adequacy assessment or final misdiagnosis.Conclusions:These data show that celiac ganglia may be infrequently encountered, especially with intra-abdominal EUS-guided FNA targeting nodes or masses near the celiac region. Therefore, cytologists should be aware of the possibility of finding ganglionic cells in EUS-guided FNA samples.

Issue Highlights

Issue Highlights

The diagnostic value of endoscopic ultrasound-guided fine-needle aspiration biopsy of cell block with immunostaining for pancreatic lesions

Objective To evaluate the diagnostic value of the cell block （CB） with immunostaining method by endoscopic ultrasound-guided fine-needle aspiration （EUS-FNA） biopsy for pancreatic lesions. Methods A total of 72 patients with pancreatic lesions underwent EUS-FNA at the First Affiliated Hospital of Guangxi Medical University from March 2012 to June 2013. The EUS-FNA samples of all patients were processed by conventional smear cytology, liquid-based cytology （LBC） and the cell block with immunos- taining. Results There were 61 pancreatic patients who were finally diagnosed as having pancreatic tumors, including 55 cases of pancreatic cancer, 2 pancreatic solid pseudopapillary tumor, 4 pancreatic endocrine tumors （PETs） , and 11 benign lesions： 4 chronic pancreatitis, 2 pancreatic tuberculosis ,4 pancreatitis and 1 pancreatic mucinous cystadenoma. The diagnostic sensitivity of conventional smear cytology, liquid-based cytology and cell block with immuno-staining method were 68. 9% （42/61）, 75.4% （46/61） and 90. 2% （ 55/61 ） , respectively. The diagnostic specificity of three methods were all 100.0%. The diagnostic accura- cy rates were 73.6% （53/72） ,79. 2% （57/72） and 91.7% （66/72）, respectively. The diagnostic accuracy rate of the cell block with immunostaining was higher than those of conventional smear cytology （ P 〈 0. 05） and the liquid-based cytology （P 〈 0. 05 ）. Conclusion Endoscopic ultrasound-guided fine-needle aspiration is a safe and effective method with high sensitivity and specificity in the diagnosis of pancreatic lesions. Cell block method combining immunohistochemistry helps to increase the diagnosis and histological diagnosis of pancreatic lesions. The cell block has a greater clinical value in the diagnosis of pancreatic lesions. Key words: Endosonography; Fine needle aspiration; Liquid-based cytology; Cell block ; Pancreatic lesions

ZIP4 is a Novel Diagnostic and Prognostic Marker in Human Pancreatic Cancer: A Systemic Comparison Between EUS-FNA and Surgical Specimens

Aberrant expression of a zinc transporter ZIP4 in pancreatic ductal adenocarcinoma (PDAC) has been shown to contribute to tumor progression and is a potential target for individualized therapy. The overall objective of this study was to determine whether ZIP4 could serve as a novel diagnostic and prognostic marker in human PDAC, and if it can be assessed by minimally invasive sampling using endoscopic ultrasound guided fine needle aspiration (EUS-FNA). Immunohistochemistry was performed to compare ZIP4 expression in the PDAC samples obtained from EUS-FNA and matched surgical tumors (parallel control). Samples were reported by sensitivity, specificity, and predictive values, all with 95% confidence intervals (CI). A total of 23 cases with both FNA and surgical specimens were evaluated. We found that ZIP4 was significantly overexpressed in tumor cells from both sets of samples. The sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) of ZIP4 for the diagnosis of PDAC were 72.9%, 72.5%, 76.1%, and 69.0% in EUS-FNA samples, and were 97.9%, 65.4%, 83.9%, and 94.4% in surgical specimens, respectively. The association between the positive rate of ZIP4 expression in FNA and surgical samples is statistically significant (P=0.0216). Both the intensity and percentage of ZIP4 positive cells from the surgical samples correlated significantly with tumor stage (P=0.0025 and P=0.0002). ZIP4 intensity level in FNA samples was significantly associated with tumor differentiation and patient survival. These results indicate that EUS-FNA is capable of non-operative detection of ZIP4, thus offering the potential to direct pre-operative detection and targeted therapy of PDAC.

A MicroRNA-Based Test Improves Endoscopic Ultrasound–Guided Cytologic Diagnosis of Pancreatic Cancer

Endoscopic ultrasound-guided fine-needle aspiration (EUS-FNA) in combination with cytopathology is the optimal method for diagnosis and staging of pancreatic ductal adenocarcinoma (PDAC) and other pancreatic lesions. Its clinical utility, however, can be limited by high rates of indeterminate or false-negative results. We aimed to develop and validate a microRNA (miRNA)-based test to improve preoperative detection of PDAC. Levels of miRNAs were analyzed in a centralized clinical laboratory by relative quantitative polymerase chain reaction in 95 formalin-fixed paraffin-embedded specimens and 228 samples collected by EUS-FNA during routine evaluations of patients with solid pancreatic masses at 4 institutions in the United States, 1 in Canada, and 1 in Poland. We developed a 5-miRNA expression classifier, consisting of MIR24, MIR130B, MIR135B, MIR148A, and MIR196, that could identify PDAC in well-characterized, formalin-fixed, paraffin-embedded specimens. Detection of PDAC in EUS-FNA samples increased from 78.8% by cytology analysis alone (95% confidence interval, 72.2%-84.5%) to 90.8% when combined with miRNA analysis (95% confidence interval, 85.6%-94.5%). The miRNA classifier correctly identified 22 additional true PDAC cases among 39 samples initially classified as benign, indeterminate, or nondiagnostic by cytology. Cytology and miRNA test results each were associated significantly with PDAC (P < .001), with positive predictive values greater than 99% (95% confidence interval, 96%-100%). We developed and validated a 5-miRNA classifier that can accurately predict which preoperative pancreatic EUS-FNA specimens contain PDAC. This test might aid in the diagnosis of pancreatic cancer by reducing the number of FNAs without a definitive adenocarcinoma diagnosis, thereby reducing the number of repeat EUS-FNA procedures.

Comparison of 22-gauge aspiration needle with 22-gauge biopsy needle in endoscopic ultrasonography-guided subepithelial tumor sampling

Objective. Endoscopic ultrasonography (EUS)-guided fine-needle aspiration (EUS-FNA) may facilitate tissue sampling for histopathological diagnosis of subepithelial tumors (SETs) in the gastrointestinal (GI) tract. However, immunohistochemistry is not always feasible using EUS-FNA samples due to the low quality of specimens often obtained by aspiration. This study aimed to compare the use of 22-gauge (G) EUS-guided fine-needle biopsy (EUS-FNB) with 22G EUS-FNA for core sampling used for histopathological examination, including immunohistochemistry, in patients with GI SETs. Methods.Twenty-eight patients with GI SETs ≥2 cm in size were prospectively enrolled at five university hospitals in Korea between January and June 2013. They were randomized to undergo either EUS-FNB or EUS-FNA. Results. A total of 22 patients was finally analyzed in this study: 10 and 12 patients underwent EUS-FNA and EUS-FNB, respectively. Compared to the EUS-FNA group, the EUS-FNB group had a significantly lower median number of needle passes to obtain macroscopically optimal core samples (4 vs. 2, p = 0.025); higher yield rates of macroscopically and histologically optimal core samples with three needle passes (30% vs. 92%, p = 0.006; 20% vs. 75%, p = 0.010, respectively); and a higher diagnostic sufficiency rate (20% vs. 75%, p = 0.010). No technical difficulties were encountered in either group. Conclusions.This study shows that EUS-FNB has a better ability to obtain histological core samples and a higher diagnostic sufficiency rate than EUS-FNA and that EUS-FNB is a feasible, safe, and preferable modality for adequate core sampling for histopathological diagnosis of GI SETs.

Abstract A39: Analysis of endoscopic ultrasound (EUS) - guided fine needle aspiration (FNA) samples from patients with suspected pancreatic ductal adenocarcinomas (PDACS) using plectin-1 (PLEC1) and KRAS oncogene mutation analysis.

Abstract Introduction: Pancreatic cancer remains an incurable and rapidly lethal cancer, with a 5-year survival rate of less than 10%. Little to nothing is known about advanced pancreatic cancer because of problems with tissue availability and usually insufficient quantity of tissue available for diagnosis and study. One of the key features of genetic basis of PDAC is the point mutation of KRAS oncogene occurred in 95 % of its pathogenesis. Plec1 was also recently identified as PDAC specific biomarker. Aims and Methods: The aim of this study was to find out the techniques using minimal specimens in order to achieve accurate diagnosis of patients with suspected PDACs. DNA was extracted from EUS-guided FNA samples in 80 patients with suspected PDACs. Seven different somatic mutations of KRAS oncogene were evaluated using ARMS and Scorpions technology, and immunohistochemical staining of Plec1 antibody was done. Results: The total of 80 study patients with suspected of PDACs underwent EUS-guided FNA and the final diagnosis were as follows; PDAC: 63 patients, pancreas neuroendocrine tumor: 4, autoimmune pancreatitis: 3, chronic pancreatitis: 1, metastasis to pancreas: 6, others: 3. The sensitivity and specificity of pathologic diagnosis in EUS-guided FNA samples were 87% and 88% respectively. When we combine KRAS oncogene mutational analysis and pathologic reports of FNA samples, we could get the following sensitivities and specificities; 95% vs. 97%. Also, adding the results of plectin-1 antibodies of immunohistochemical staining could increase the sensitivity of PDAC diagnosis. Conclusion: Triple combinations of the techniques (pathologic reports, KRAS oncogene mutational analysis, Plec1 staining) could increase accuracy, however, cost-effectiveness and laborious work should be taken into consideration in clinical practice. Further investigation of using minimal specimens to study PDACs may enable us to understand biological behavior and to identify better tools for the diagnosis and treatment of this deadly cancer. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):A39. Citation Format: Joo Kyung Park, Byung Jun Song, Ji Kon Ryu, Yong-Tae Kim, Se-Hoon Lee. Analysis of endoscopic ultrasound (EUS) - guided fine needle aspiration (FNA) samples from patients with suspected pancreatic ductal adenocarcinomas (PDACS) using plectin-1 (PLEC1) and KRAS oncogene mutation analysis. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr A39.

Grading epithelial atypia in endoscopic ultrasound‐guided fine‐needle aspiration of intraductal papillary mucinous neoplasms: An international interobserver concordance study

Preoperative cytological analysis of cyst fluid from intraductal papillary mucinous neoplasms (IPMN) contributes to risk stratification for malignancy. To the authors' knowledge, agreement among pathologists in grading epithelial atypia in IPMN cyst fluids has not been studied to date. Blinded to the histological grade, 4 observers independently graded 60 cell groups from endoscopic ultrasound-guided fine-needle aspiration samples of 24 IPMNs of various grades as either 0 (nonlesional), 1 (low-grade dysplasia), 2 (intermediate-grade dysplasia [IGD]), 3 (high-grade dysplasia), or 4 (invasive adenocarcinoma). The percentage of agreement and the kappa (k) coefficient were calculated for these 5 tiers and a reduced 2-tier grouping (low-grade [LG] [0-1 and 0-2] vs high-grade [HG] [2-4 and 3-4]). Agreement between the 2 most experienced reviewers established a consensus diagnosis that was compared with the histological grade. Among the 4 reviewers there was poor agreement using a 5-tiered system and fair agreement with the 2-tiered system. LG and HG grouping of IGD did not appear to greatly affect agreement, with 54% agreement for grouping 0 to 2 and 3 to 4 (k = 0.45) and 52% agreement for grouping 0 to 1 and 2 to 4 (k = 0.44). The 2 most experienced reviewers had 87% agreement for the 0 to 2 and 3 to 4 grouping (k = 0.74) and 88% for the 0 to 1 and 2 to 4 grouping (k = 0.71). HG atypia on cytology with IGD grouped as LG yielded a sensitivity of 82%, with a specificity of 70% for detecting a cyst with HG morphology. Grading cellular atypia in cyst fluids requires experience, which results in very good interobserver agreement and good correlation with histology using a 2-tiered LG and HG grading system and IGD classified as LG.

Immunohistochemical analysis of E‐cadherin and zeste homolog 2 expression in endoscopic ultrasound‐guided fine‐needle aspiration of pancreatic adenocarcinoma

Recent studies have demonstrated that partial or complete loss of E-cadherin (EC) and nuclear accumulation of zeste homolog 2 (EZH2) are hallmarks of poorly differentiated pancreatic adenocarcinoma (PAC). Depletion of EZH2 sensitizes cancer cells to chemotherapy in vitro. The objective of this study was to determine EC and EZH2 expression by immunohistochemistry (IHC) in samples obtained by endoscopic ultrasound-guided fine-needle aspiration (EUS-FNA) as potential biomarkers for treatment and disease prognosis. Thirty-eight EUS-FNA samples from patients with a PAC diagnosis were analyzed by IHC for EC and EZH2 expression. Seven corresponding surgical resection specimens were included in the study. The intensity of EZH2 and EC expression in PAC and in normal gastrointestinal pick-ups (internal positive control) was scored by using a 4-tier grading system. EC demonstrated a focal, weak-to-moderate decrease in 24 PAC samples. Complete loss of EC expression was observed in the poorly differentiated areas represented by single tumor cells. The average staining intensity of EC in samples of poorly differentiated PAC was significantly lower than that of moderately differentiated PAC samples (P = .0005). EZH2 was variably positive in 31 of 38 PAC samples. The average staining intensity of EZH2 in moderately and poorly differentiated PACs did not differ significantly (P = .81). EC and EZH2 expression was determined reliably by IHC on paraffin sections of EUS-FNA cell block specimens. The current results were consistent with prior reports indicating a decrease or loss of EC expression in poorly differentiated PAC. However, EZH2 expression did not always correlate inversely with EC expression and was more heterogeneous.

Analyzing S100A6 Expression in Endoscopic Ultrasonography-guided Fine-needle Aspiration Specimens

Endoscopic ultrasonography-guided fine-needle aspiration (EUS-FNA) cytology in combination with other tests is necessary to improve diagnostic accuracy. We evaluated the diagnostic utility of S100A6 expression in EUS-FNA tissue samples in pancreatic ductal adenocarcinoma (PDA). RNA was extracted from 36 PDA and 44 nontumor pancreatic tissues obtained during surgery. S100A6 expression was quantified by real-time reverse transcription-polymerase chain reaction, and receiver operating characteristic analysis was performed to determine the cutoff value for PDA. We preoperatively performed EUS-FNA in 52 patients with pancreatic masses, then prospectively evaluated the diagnostic value of S100A6 expression of EUS-FNA samples in pancreatic cancer diagnosis. S100A6 immunohistology was conducted to validate the S100A6 expression data in PDA samples. Of the 52 EUS-FNA patients with pancreatic masses, RNA was successfully extracted from 44, which comprised 34 pancreatic cancer patients and 10 patients with benign pancreatic diseases. Cytology results were malignant in 23 cases, benign in 9, and atypical or abnormal in 12. The sensitivity, specificity, and accuracy of cytology for diagnosis of pancreatic cancer were 67.65%, 100%, and 75%, respectively. When an S100A6 expression >0.005248 was defined as positive for malignancy, the sensitivity, specificity, and accuracy of S100A6 expression of EUS-FNA for diagnosis of pancreatic cancer were 88.24%, 90.00%, and 88.64%, respectively. Quantification of S100A6 expression in EUS-FNA samples had a high sensitivity and specificity for the diagnosis of PDA.

Comparison of Surgical and Endoscopic Sample Collection for Pancreatic Cyst Fluid Biomarker Identification

Significant efforts are underway to develop new biomarkers from pancreatic cyst fluid. Previous research has made use of cyst fluid collected from surgically removed cysts, but the clinical implementation of biomarkers would use cyst fluid collected by endoscopic ultrasound-guided, fine-needle aspiration (EUS-FNA). The purpose of this study was to investigate the clinical applicability of cyst fluid research obtained using surgical specimens. Matched pairs of operating-room collected (OR) and EUS-FNA samples from 12 patients were evaluated for the levels of three previously described biomarkers, CA 19-9, CEA, and glycan levels detected by wheat germ agglutinin on MUC5AC (MUC5AC-WGA). CA 19-9 and MUC5AC-WGA correlated well between the sample types, although CEA was more variable between the sample types for certain patients. The variability was not due to the time delay between EUS-FNA and OR collection or differences in total protein concentrations but may be caused by contamination of the cyst fluid with blood proteins. The classification of each patient based on thresholds for each marker was perfectly consistent between sample types for CA 19-9 and MUC5AC-WGA and mostly consistent for CEA. Therefore, results obtained using OR-collected pancreatic cyst fluid samples should reliably transfer to the clinical setting using EUS-FNA samples.

Endoscopic ultrasonography-guided fine needle aspiration: Relatively low sensitivity in the endosonographer population

To assess the characteristics and quality of endoscopic ultrasonography-guided fine needle aspiration (EUS-FNA) in a large panel of endosonographers. A survey was conducted during the 13th annual live course of endoscopic ultrasonography (EUS) held in Amsterdam, Netherlands. A 2-page questionnaire was developed for the study. Content validity of the questionnaire was determined based on input by experts in the field and a review of the relevant literature. It contained 30 questions that pertained to demographics and the current practice for EUS-FNA of responders, including sampling technique, sample processing, cytopathological diagnosis and sensitivity of EUS-FNA for the diagnosis of solid mass lesions. One hundred and sixty-one endosonographers who attended the course were asked to answer the survey. This allowed assessing the current practice of EUS-FNA as well as the self-reported sensitivity of EUS-FNA for the diagnosis of solid mass lesions. We also examined which factors were associated with a self-reported sensitivity of EUS-FNA for the diagnosis of solid mass lesions > 80%. Completed surveys were collected from 92 (57.1%) of 161 endosonographers who attended the conference. The endosonographers had been practicing endoscopy and EUS for 12.5 ± 7.8 years and 4.8 ± 4.1 years, respectively; one third of them worked in a hospital with an annual caseload > 100 EUS-FNA. Endoscopy practices were located in 29 countries, including 13 countries in Western Europe that totaled 75.3% of the responses. Only one third of endosonographers reported a sensitivity for the diagnosis of solid mass lesions > 80% (interquartile range of sensitivities, 25.0%-75.0%). Factors independently associated with a sensitivity > 80% were (1) > 7 needle passes for pancreatic lesions or rapid on-site cytopathological evaluation (ROSE) (P < 0.0001), (2) a high annual hospital caseload (P = 0.024) and (3) routine isolation of microcores from EUS-FNA samples (P = 0.042). ROSE was routinely available to 27.9% of respondents. For lymph nodes and pancreatic masses, a maximum of three needle passes was performed by approximately two thirds of those who did not have ROSE. Microcores were routinely harvested from EUS-FNA samples by approximately one third (37.2%) of survey respondents. EUS-FNA sensitivity was considerably lower than reported in the literature. Low EUS-FNA sensitivity was associated with unavailability of ROSE, few needle passes, absence of microcore isolation and low hospital caseload.

Molecular Biologic Approach to the Diagnosis of Pancreatic Carcinoma Using Specimens Obtained by EUS-Guided Fine Needle Aspiration

We review the utility of endoscopic ultrasound-guided fine needle aspiration (EUS-FNA), a rapid, safe, cost-effective, and accurate diagnostic modality for evaluating pancreatic tumors. EUS-FNA is currently used for the diagnosis and staging of pancreatic tumors. The sensitivity of EUS-FNA for pancreatic malignancy ranges from 75% to 94%, and its specificity approaches 100% in most studies. However, EUS-FNA has some limitations in the diagnosis of well-differentiated or early-stage cancers. Recent evidence suggests that molecular biological analysis using specimens obtained by EUS-FNA improves diagnostic sensitivity and specificity, especially in borderline cytological cases. It was also reported that additional information regarding patient response to chemotherapy, surgical resectability, time to metastasis, and overall survival was acquired from the genetic analysis of specimens obtained by EUS-FNA. Other studies have revealed that the analysis of KRAS, MUC, p53, p16, S100P, SMAD4, and microRNAs is helpful in making the diagnosis of pancreatic carcinoma. In this paper, we describe the present state of genetic diagnostic techniques for use with EUS-FNA samples in pancreatic diseases. We also discuss the role of molecular biological analyses for the diagnosis of pancreatic carcinoma.