EUS-FNA Samples Research Articles

Abstract B110: Molecular subtypes from endoscopy ultrasound-guided biopsy samples collected at initial diagnosis predict therapeutic response to neoadjuvant chemotherapy in pancreatic cancer

Abstract Introduction Pancreatic cancer has a poor prognosis, despite the availability of various therapeutic approaches. Recent evidence suggests that precision strategies tailored to the molecular profiles of patients’ tumors can improve oncologic outcomes in solid cancers. This study aimed to identify clinically relevant molecular subtypes based on transcriptomic data of samples collected through ultrasound-guided fine needle aspiration biopsies (EUS-FNA) performed before neoadjuvant chemotherapy (NACT) in patients with borderline resectable pancreatic cancer (BRPC) or locally advanced pancreatic cancer (LAPC). Methods Total RNA sequencing was performed on tumor samples collected using EUS-FNA prior to NACT. The response to NACT as well as the clinicopathologic results of patients were recorded. To determine molecular subtypes based on transcriptomic data, an unbiased approach based on non-negative matrix factorization (NMF) was utilized. Gene set enrichment analysis (GSEA) and ingenuity pathway analysis (IPA) were performed to describe the molecular characteristics of subgroups. Clinicopathologic and genetic characteristics of subgroups were validated using a published cohort database, the cancer genome atlas for pancreatic cancer (TCGA-PAAD). In addition, spatial transcriptomic profiling was conducted to investigate the interactive molecular characteristics the tumor microenvironments specific to each subtype. Results Sequencing data was obtained from samples collected from 35 patients. Two molecular subtypes were identified. GSEA showed that subgroup 2 enhanced expression of gene sets pertaining to G2M checkpoint, E2F targets, KRAS signaling, and mitotic spindle, all of which are related to cancer-specific proliferation. IPA results revealed the subtype-specific upregulation of WNT11 and ASCL1, which are implicated in the aggressive behavior of pancreatic cancer in this subtype. Notably, patients in subtype 2, characterized by aggressive cancer behavior, exhibited poorer outcomes following neoadjuvant treatment than those in subtype 1. Similar results were found in the TCGA-PAAD cohort. Using spatial transcriptomic analysis, we revealed that a set of genes representing subtype 2 were highly expressed in a tumor area of a patient belonging to subtype 2, but showed low expression in a patient belonging to subtype 1. Conclusion Two molecular subtypes defined based on transcriptomic data from EUS-FNA samples obtained during the initial diagnosis of pancreatic cancer possessed distinct molecular characteristics and were related to different clinical outcomes, especially regarding the therapeutic efficacy of neoadjuvant treatment. These results demonstrate that, following robust validation with corroborative evidence, precision strategies based on tumor biology should be provided in a clinical setting to improve treatment outcomes. Citation Format: Woohyung Lee. Molecular subtypes from endoscopy ultrasound-guided biopsy samples collected at initial diagnosis predict therapeutic response to neoadjuvant chemotherapy in pancreatic cancer [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Pancreatic Cancer; 2023 Sep 27-30; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(2 Suppl):Abstract nr B110.

Accurate non-invasive grading of nonfunctional pancreatic neuroendocrine tumors with a CT derived radiomics signature

PurposeThe purpose of this study was to develop a radiomics-signature using computed tomography (CT) data for the preoperative prediction of grade of nonfunctional pancreatic neuroendocrine tumors (NF-PNETs). Materials and methodsA retrospective study was performed on patients undergoing resection for NF-PNETs between 2010 and 2019. A total of 2436 radiomic features were extracted from arterial and venous phases of pancreas-protocol CT examinations. Radiomic features that were associated with final pathologic grade observed in the surgical specimens were subjected to joint mutual information maximization for hierarchical feature selection and the development of the radiomic-signature. Youden-index was used to identify optimal cutoff for determining tumor grade. A random forest prediction model was trained and validated internally. The performance of this tool in predicting tumor grade was compared to that of EUS-FNA sampling that was used as the standard of reference. ResultsA total of 270 patients were included and a fusion radiomic-signature based on 10 selected features was developed using the development cohort (n = 201). There were 149 men and 121 women with a mean age of 59.4 ± 12.3 (standard deviation) years (range: 23.3–85.0 years). Upon internal validation in a new set of 69 patients, a strong discrimination was observed with an area under the curve (AUC) of 0.80 (95% confidence interval [CI]: 0.71–0.90) with corresponding sensitivity and specificity of 87.5% (95% CI: 79.7–95.3) and 73.3% (95% CI: 62.9–83.8) respectively. Of the study population, 143 patients (52.9%) underwent EUS-FNA. Biopsies were non-diagnostic in 26 patients (18.2%) and could not be graded due to insufficient sample in 42 patients (29.4%). In the cohort of 75 patients (52.4%) in whom biopsies were graded the radiomic-signature demonstrated not different AUC as compared to EUS-FNA (AUC: 0.69 vs. 0.67; P = 0.723), however greater sensitivity (i.e., ability to accurately identify G2/3 lesion was observed (80.8% vs. 42.3%; P < 0.001). ConclusionNon-invasive assessment of tumor grade in patients with PNETs using the proposed radiomic-signature demonstrated high accuracy. Prospective validation and optimization could overcome the commonly experienced diagnostic uncertainty in the assessment of tumor grade in patients with PNETs and could facilitate clinical decision-making.

Targeted transcriptomic analysis of pancreatic adenocarcinoma in EUS-FNA samples by NanoString technology.

Background: Integration of transcriptomic testing into EUS-FNA samples is a growing need for precision oncology in pancreatic ductal adenocarcinoma (PDAC). The NanoString platform is suitable for transcriptome profiling in low yield RNA samples. Methods: Inclusion of patients that underwent EUS-FNA cytological diagnosis of pancreatic ductal adenocarcinoma using 19G and/or 22G needles and subsequent surgical resection. Formalin-fixed, paraffin-embedded (FFPE) cytological and surgical samples underwent RNA extraction and transcriptomic analysis using a custom 52-gene NanoString panel of stromal PDAC features. Cell type abundance was quantified in FFPE specimens and correlated. Results: 18 PDAC patients were included. Mean EUS-FNA passes was 2 + 0.7. All FFPE passed the RNA quality control for genomic analysis. Hierarchical clustering on the global gene expression data showed that genes were differentially expressed between EUS and surgical samples. A more enriched cancer-associated fibroblasts and epithelial-mesenchymal transition transcriptomic profile was observed across surgical specimens whereas immunological biomarkers were more represented in EUS-FNA samples. Cytological examination confirmed a scanty representation of CAF and more immunological cell abundance in cytological samples in comparison to surgical specimens. Conclusion: Targeted transcriptomic NanoString profiling of PDAC samples obtained by EUS-FNA is a feasible approach for pre-surgical molecular analysis although stromal CAF/EMT mRNA biomarkers are underrepresented.

Risk of Malignancy Using the Diagnostic Categories Proposed by the World Health Organization International System for Reporting Pancreaticobiliary Cytopathology

Background: The World Health Organization (WHO) proposed an updated reporting system for pancreaticobiliary cytology, which moves low-grade malignancies to “positive for malignancy” group and serous cystadenoma to “negative for malignancy” group. The WHO system also created two new categories, namely, pancreatic neoplasia-low grade (PaN-Low) and pancreatic neoplasia-high grade (PaN-High), which includes neoplastic mucinous cysts and stratifies them according to their cytologic atypia. The risk of malignancy (ROM) of the new categories of the WHO system needs to be defined. Methods: Cytologic slides of all patients, who underwent endoscopic ultrasound (EUS)-guided fine needle aspiration (FNA) biopsy at our institution from January 2010 to December 2021 and had a histopathological or clinical follow-up of at least 6 months, were reviewed and reclassified under the Papanicolaou Society of Cytopathology (PSC) and WHO reporting systems. The absolute ROM was calculated for each category of both reporting systems. Results: A total of 420 EUS-FNA samples from 410 patients were reviewed and reclassified. The absolute ROM for the proposed WHO system was 35% for “nondiagnostic,” 1.0% for “negative for malignancy,” 69.0% for “atypical,” 11% for “PaN-Low,” 100% for “PaN-High,” 91% for “suspicious for malignancy,” and 100% for “malignant.” Comparatively, the absolute ROM under the PSC reporting system was 34% for “nondiagnostic,” 1.0% for negative (for malignancy), 50.0% for “atypical,” 0.0% for “neoplastic: benign,” 16% for “neoplastic: other,” 88% for “suspicious for malignancy,” and 100% for “positive or malignant.” Conclusion: The proposed WHO international reporting system has advantages regarding risk stratification improvement and case management.

The EUS molecular evaluation of pancreatic cancer: A prospective multicenter cohort trial.

Background and Objectives:Patients with locally advanced or metastatic pancreatic ductal adenocarcinoma (A-PDAC) are not candidates for surgical resection and are often offered palliative chemotherapy. The ready availability of a safe and effective tumor sampling technique to provide material for both diagnosis and comprehensive genetic profiling is critical for informing precision medicine in A-PDAC, thus potentially increasing survival. The aim of this study is to examine the feasibility and benefits of routine comprehensive genomic profiling (CGP) of A-PDAC using EUS-FNA material.Methods:This is a prospective cohort study to test the clinical utility of fresh frozen or archival EUS-FNA samples in providing genetic material for CGP. The results of the CGP will be reviewed at a molecular tumor board. The proportion of participants that have a change in their treatment recommendations based on their individual genomic profiling will be assessed. Correlations between CGP and stage, prognosis, response to treatment and overall survival will also be investigated. This study will open to recruitment in 2020, with a target accrual of 150 A-PDAC patients within 36 months, with a 2-year follow-up. It is expected that the majority of participants will be those who have already consented for their tissue to be biobanked in the Victorian Pancreatic Cancer Biobank at the time of diagnostic EUS-FNA. Patients without archival or biobanked material that is suitable for CGP may be offered a EUS-FNA procedure for the purposes of obtaining fresh frozen material.Discussion:This trial is expected to provide crucial data regarding the feasibility of routine CGP of A-PDAC using EUS-FNA material. It will also provide important information about the impact of this methodology on patients’ survival.

Diagnostic value of digital droplet polymerase chain reaction and digital multiplexed detection of single-nucleotide variants in pancreatic cytology specimens collected by EUS-guided FNA

EUS-guided FNA is recommended as a first-line procedure for the histopathologic diagnosis of pancreatic cancer. Molecular analysis of EUS-FNA samples might be used as an auxiliary tool to strengthen the diagnosis. The current study aimed to evaluate the diagnostic performances of K-ras testing using droplet digital polymerase chain reaction (ddPCR) and a novel single-nucleotide variant (SNV) assay performed on pancreatic EUS-FNA samples. EUS-FNA specimens from 31 patients with pancreatic masses (22 pancreatic ductal adenocarcinomas, 7 chronic pancreatitis, and 2 pancreatic neuroendocrine tumors) were included in the study. K-ras testing was initially performed by ddPCR. In addition, mutational status was evaluated using an SNV assay by NanoString technology, using digital enumeration of unique barcoded probes to detect 97 SNVs from 24 genes of clinical significance. The overall specificity and sensitivity of cytologic examination were 100% and 63%, respectively. K-ras mutation testing was performed using ddPCR, and the sensitivity increased to 87% with specificity 90%. The SNV assay detected at least 1 variant in 90% of pancreatic ductal adenocarcinoma samples; the test was able to detect 2 K-ras codon 61 mutations in 2 cases of pancreatic ductal adenocarcinoma, which were missed by ddPCR. The overall diagnostic accuracy of the cytologic examination alone was 74%, and it increased to 91% when the results of both molecular tests were considered for the cases with negative and inconclusive results. The current study illustrated that integration of K-ras analysis with cytologic evaluation, especially in inconclusive cases, can enhance the diagnostic accuracy of EUS-FNA for pancreatic lesions.

Evaluation of Ki67 Index in Endoscopic Ultrasound-Guided Fine Needle Aspiration Samples for the Assessment of Malignancy Risk in Gastric Gastrointestinal Stromal Tumors

Background: The risk of malignancy in resected gastrointestinal stromal tumors (GISTs) depends on tumor size, location, and mitotic index. Reportedly, the Ki67 index has a prognostic value in resected GISTs. We aimed to analyze the accuracy of endoscopic ultrasound (EUS)-guided fine needle aspiration (FNA) samples with reference to Ki67 index, using surgical specimens as the gold standard. Methods: Fifty-five patients who underwent EUS-FNA followed by surgical resection for gastric GISTs were retrospectively analyzed. Patients’ age and sex; tumors’ size and location; mitotic index, cell type, cellularity, pleomorphism, presence of ulceration, hemorrhage, necrosis, mucosal or serosal invasion, growth pattern, and Ki67 index based on pathology were investigated. Results: Location in fundus, ulceration, hemorrhage, mucosal invasion, and Ki67 index in surgical specimens were significant in predicting high-risk groups (p < 0.05) on univariate analysis. Frequency of bleeding (p = 0.034) and the Ki67 index (p = 0.018) were the only independent significant factors in multivariate analysis. The optimal cutoff level of Ki67 was 5%, with 88.2% sensitivity and 52.8% specificity (p = 0.021). The mean Ki67 index was lower in EUS-FNA samples than in surgical specimens (2% [1–15] versus 10% [1–70], p = 0.001). The rank correlation coefficient value of Ki67 was 0.199 (p = 0.362) between EUS-FNA and surgical samples and showed no reliability for EUS-FNA samples. Conclusion: The Ki67 index in resected specimens correlated with high-risk GISTs, although it had no additive value to the current criteria. The Ki67 index in EUS-guided FNA samples is not a reliable marker of proliferation in GISTs.

Mutation analysis of gastrointestinal stromal tumors using RNA obtained via endoscopic ultrasound-guided fine-needle aspiration.

Endoscopic ultrasound-guided fine-needle aspiration biopsy (EUS-FNA) is useful for pathologically diagnosing gastrointestinal stromal tumor (GIST) before surgery. However, its role in mutation analysis remains unclear. To examine the feasibility of analyzing GIST mutations using mRNA obtained with EUS-FNA, we prospectively enrolled 41 patients with subepithelial lesion from which EUS-FNA was successfully acquired tissue sample. Thirty-two, 5, and 4 subepithelial lesions were diagnosed as GISTs, schwannomas, and leiomyomas, respectively. After RNA was extracted from FNA sample, RNA was converted to cDNA. Full-length sequence of the KIT cDNA amplified via the polymerase chain reaction (PCR) was successful in 31 (96.9%) out of 32 GIST and three out of 9 non-GIST (33.3%). The KIT mutation statuses of 31 GISTs in which KIT cDNA was amplified were successfully determined through directional sequencing. Furthermore, 15 of 16 surgically excised GISTs exhibited the same mutation status in both the EUS-FNA and resected samples. In vitro experiment, the minimum number of cells required to amplify full-length of KIT cDNA from RNA was one-tenth of that required to amplify KIT exon11 gene from DNA. This study clarifies that mutation analysis using RNA obtained with EUS-FNA is feasible and reliable. Moreover, our data would support that RNA-based mutation is superior to DNA-based mutation analysis in GIST.

Sa1360 – Heat Shock Protein 27(HSP27) Expression in Eus-Fna Samples for Gem Sensitivity Prediction in Pancreatic Cancer

Sa1360 – Heat Shock Protein 27(HSP27) Expression in Eus-Fna Samples for Gem Sensitivity Prediction in Pancreatic Cancer

Mutational landscape of pancreatic cancer and genomic analysis using EUS-FNA samples

Mutational landscape of pancreatic cancer and genomic analysis using EUS-FNA samples

EUS-Guided FNA Biopsy of Solid Pancreatic Lesions: A Review of 111 Cases and Comparative Study of Diff-Quik/PAP/Thin Prep Staining Techniques

Background: Pancreatic cancer is the fourth leading cause of cancer associated deaths in the United States and ranks ninth in its incidence. EUS-guided FNA cytology has emerged as an important diagnostic tool since Vilman and Grimm (1992) made the first reports of endoscopic guided fine needle aspiration. Currently, EUS-guided FNA biopsy of the pancreas is a standard practice for the diagnosis and staging of pancreatic malignancy. Aim: The current study was carried out with the following objectives. 1. To find out the diagnostic accuracy, sensitivity, and specificity of EUS-guided FNA of the pancreatic malignancy by correlating cytologic diagnosis with histologic diagnosis and other investigations. 2. To know the diagnostic accuracy of air-dried smears using Diff-Quik Stain. 3. To compare the diagnosis obtained by Diff-Quik, conventional PAP Stain smears/cytospin preparation and Thin Prep (liquid-based media). Methods: 111 cases of EUS-guided FNA biopsy of the pancreas performed during the year January 2008 to December 2009 having solid pancreatic mass/lesion on the USG/CT and suspicious for the malignancy or malignant on clinical and radiologic investigations were reviewed in August 2011 at the Department of Pathology and Laboratory Medicine, Hospital of University of Pennsylvania. Results: There was an 83.9% correlation between Diff-Quick diagnosis and the final cytologic diagnosis. The overall diagnostic accuracy for the malignancy was 89.7%, sensitivity 90.6%, and specificity 100% of the cases where the cytologic diagnosis was correlated with histologic diagnosis and the other investigations. The positive predictive value for the malignancy was 100%. The false negative diagnosis was encountered in 10.3% cases. 51% of the cases showed intranuclear vacuoles, in the malignant cases on Diff-Quik stain. Conclusion: EUS FNA of the pancreas is a safe and reliable technique with high diagnostic accuracy, sensitivity, and specificity. The Diff-Quik stain smear is a useful technique for the rapid on-site cytologic evaluation for the detection of malignancy of the pancreas. Thin Prep was found superior to the PAP/Diff-Quik stain for the diagnosis of the malignancy. The EUS FNA samples processed by multiple staining techniques help to improve the diagnostic accuracy and sensitivity.

Sa1372 - Molecular Markers Performed on EUS-FNA Samples in Assessing Diagnosis of Pancreatic Cancer: A Systematic Review and Meta-Analysis

Sa1372 - Molecular Markers Performed on EUS-FNA Samples in Assessing Diagnosis of Pancreatic Cancer: A Systematic Review and Meta-Analysis

Diagnostic Yield of Micro Forceps Biopsies during Endoscopic Ultrasound Evaluation of Pancreatic Cystic Lesions

Introduction: Evaluation of pancreatic cystic lesions (PCLs) can be challenging and requires a combination of radiologic imaging, morphologic characteristics on endoscopic ultrasound (EUS), cyst fluid cytology, and cyst fluid analysis. Through-the-needle micro forceps (Moray micro forceps, US Endoscopy, Mentor, OH) are a recent addition to EUS armamentarium. These forceps allow tissue sampling from PCLs and may improve diagnostic yield. To date, scant data exists on the use of micro forceps biopsy for these lesions. The main aim of this study was to assess diagnostic yield and safety of EUS-guided micro forceps biopsy for PCLs. Methods: Our electronic endoscopy database was queried to identify patients who underwent EUS-FNA of PCLs and biopsies with the micro forceps, during the same procedure. Cysts were identified on EUS and punctured using 19-G FNA needle. All patients received peri-procedural antibiotics. Cyst fluid was collected for cytologic analysis, CEA and amylase levels, and wall of the cyst was biopsied using micro forceps through the 19-G needle. Adverse events were recorded per published ASGE criteria. Results: Twenty patients (mean age 64.2 years [range 34 -87]; 8/20 male [40%]) underwent EUS-FNA and micro-forceps biopsy of PCLs from 02/2016 to 05/2017. The mean cyst size was 34.4 mm (range 13 -70 mm), with 10 cysts in the pancreatic head/uncinate and 10 in the body/tail region. Micro forceps biopsies were technically successful in all cases, and provided a diagnosis of neoplasia in 11 cases (55%), benign tissue in 8 cases (40%) and insufficient quantity in 1 case. In 4 patients (20%), micro forceps biopsy results drastically changed patient management, providing diagnoses (1 Neuroendocrine tumor, 1 mucinous cystic neoplasm, and 2 IPMNs) otherwise not suggested by cytology or cyst fluid analysis. However, cytology provided a diagnosis of mucinous neoplasm in 3 cases not detected by micro forceps biopsies. No adverse events were noted. Conclusion: Micro forceps biopsies were associated with high yield and excellent safety profile. Biopsies provided a diagnosis of neoplasia not detected by cytology in a significant number of patients but also did not identify mucinous neoplasms in a few cases as suggested by cyst fluid cytology. Hence micro forceps biopsies may be a useful adjunctive tool for EUS guided assessment of PCLs, complementing existing EUS-FNA sampling protocols.

Endoscopic ultrasound core needle for diagnosing of solid pancreatic lesions: is rapid on-site evaluation really necessary?

Endoscopic ultrasound (EUS) guided fine needle aspiration (FNA) has a crucial role in the diagnosis of solid pancreatic lesions and rapid on-site evaluation (ROSE) can increase its diagnostic yield. Rapid on-site evaluation is not always available and after EUS-FNA provides a cytology specimen with scant cellularity. Fine needle biopsy (FNB) specimens containing core tissue may theoretically overcome the limitations of EUS-FNA sampling. It could be hypothesized that EUS-FNB without ROSE could achieve the same adequacy and accuracy of EUS-FNB with ROSE. The aim of this review is to evaluate the evidence on the role of ROSE in the setting of EUS-guided tissue acquisition with core needles in patients with solid pancreatic lesions. All relevant articles were extracted up to February 2017 based on the results of searches in PubMed, Scopus and Google Scholar. A total of 21 pertinent articles were finally included. Among the included studies, 11 were performed without ROSE, 8 with ROSE, and 2 were performed both with and without ROSE. In the ROSE group we found a sensitivity, specificity, overall diagnostic adequacy and overall diagnostic accuracy of 96%, 100%, 86.5%, 85.5%, respectively, while in the no-ROSE group 86.6%, 100%, 89.5%, 86.1% were found, respectively. Mild pancreatitis (mean rates, 3.43%) and abdominal pain (mean rates, 3.6%) were reported as most frequent adverse events. Endoscopic ultrasound guided-FNB without ROSE offers similar results in terms of adequacy and accuracy as in the presence of an on-site cytopathologist.

Reliability of Ki-67 Determination in FNA Samples for Grading Pancreatic Neuroendocrine Tumors.

Neuroendocrine pancreatic tumors (PanNETs) are graded on the basis of their proliferative activity. Cytological samples are commonly the only samples available, but the determination of Ki-67 in cytology and its reliability as a measure of tumor mitotic activity is not well settled. We have retrospectively reviewed all the cases of FNA under EUS control of PanNETs in a 10-year period (2006-2016) in the Hospital Clínico San Carlos (Madrid). We identified 10 PanNET cases with histological correlation. Median age was 49.4years and the patients were mainly women. PanNETs were located more frequently in the tail of the pancreas, with a median size of 33.8mm. None of our cases was a grade 3 tumor. The seven grade 1 tumors confirmed in histology had consistent Ki-67 in cytology. In three cases (30%), there were discrepancies between the Ki-67 index measured in cytology and histology, and the differences ranged from 2 to 15%; all these cases were grade 2 tumors in histology and were graded as grade 1 tumors in FNA material. Our results are consistent with previous studies which showed understaging when tumor grade was assessed in cytological samples, mainly in G2 tumors. Previous literature has shown that Ki-67 assessment in EUS-FNA samples is a useful tool to rule out G3 tumors, but can be problematic for distinguishing G1 and G2 tumors.

Diagnosis of pancreatic lesions collected by endoscopic ultrasound-guided fine-needle aspiration using next-generation sequencing.

Endoscopic ultrasound-guided fine-needle aspiration (EUF-FNA) has improved the diagnosis of pancreatic lesions. Next-generation sequencing (NGS) facilitates the production of millions of sequences concurrently. Therefore, in the current study, to improve the detectability of oncogenic mutations in pancreatic lesions, an NGS system was used to diagnose EUS-FNA samples. A total of 38 patients with clinically diagnosed EUS-FNA specimens were analyzed; 27 patients had pancreatic ductal adenocarcinoma (PDAC) and 11 had non-PDAC lesions. DNA samples were isolated and sequenced by NGS using an Ion Personal Genome Machine system. The Cancer Hotspot Panel v2, which includes 50 cancer-related genes and 2,790 COSMIC mutations, was used. A >2% mutation frequency was defined as positive. KRAS mutations were detected in 26 of 27 PDAC aspirates (96%) and 0 of 11 non-PDAC lesions (0%). The G12, G13, and Q61 KRAS mutations were found in 25, 0, and 1 of the 27 PDAC samples, respectively. Mutations were confirmed by TaqMan® polymerase chain reaction analysis. TP53 mutations were detected in 12 of 27 PDAC aspirates (44%). SMAD4 was observed in 3 PDAC lesions and cyclin-dependent kinase inhibitor 2A in 4 PDAC lesions. Therefore, the current study was successfully able to develop an NGS assay with high clinical sensitivity for EUS-FNA samples.

Multiprobe FISH on Pancreatic Adenocarcinoma Diagnosed by EUS-FNA of the Pancreas: A Validation Study

Introduction: Cautious approach to pancreatic FNA often results in an indeterminate diagnosis on pancreatic endoscopic ultrasound guided (EUS) fine needle aspiration. Utilization of ancillary studies may help confirm a diagnosis of carcinoma of the pancreas. This has a distinct potential to help patients and clinical teams arrive at early informed patient management decisions. Recent recommendations on utilization of ancillary studies in pancreatobiliary tree analysis suggested a possible role for fluorescence in situ hybridization (FISH) studies as an adjunct to cytology for pancreatic masses (Diagn. Cytopathol. 2014; 42:351e362). Our objective for the study was to determine performance characteristics of commercially available multiprobe FISH (CEP3, CEP7, CEP17 and locus specific probe for chromosome 9p21) on EUS-FNA of the pancreas. Materials and Methods:We prospectively collected EUS-FNA samples for UroVysion (Abbott Inc) multiprobe FISH analysis. Residual FNA sample after the diagnosis was forwarded for FISH analysis in preservecyt. Sample preparation and analysis were performed as per the manufactures’ suggested protocol. A statistical analysis using a two tailed student t test was performed. Results: We analyzed 32 samples from 30 patients. The mean age was 67 yrs (range: 42-90 yrs). The tumor size ranged from 1.1 e 7 cms (mean 3.0 cms). Eighteen of the 19 (95%) adenocarcinoma were positive for UroVysion FISH. Of the 2 cases deemed negative by cytology both were considered UroVysion negative. Aneusomy was statistically significantly associated with adenocarcinoma (pZ 0.014). Of the 8 indeterminate cases 4 cases each were considered as either atypical or suspicious for adenocarcinoma. Lack of cellularity precluded adequate analysis by FISH in suspicious (nZ 3) and atypical (nZ3) cases. One of 4 (25%) cases suspicious for carcinoma was positive by FISH. Conclusion: Aneusomy on UroVysion FISH can be used as an ancillary tool to support a diagnosis of pancreatic carcinoma on EUS-FNA samples.

695: MicroRNAs for detection of pancreatic neoplasia: biomarker discovery by next generation sequencing and validation in EUS FNA and plasma samples

695: MicroRNAs for detection of pancreatic neoplasia: biomarker discovery by next generation sequencing and validation in EUS FNA and plasma samples

832 Development of a Device for Facilitating the Detection of Target Specimens From EUS-FNA Samples in Pancreatic Tumors

A 25-G needle is recommended for EUS-FNA of pancreatic tumors because fine-gauge needles have fewer complications than thicker needles and are equally useful for diagnosis (Madhoun MF, et al. Endoscopy 2013). However, it is often difficult to confirm the presence of the target specimen within a sample, since specimens collected with such fine needles are microscopic and contain blood. The use of rapid on-site evaluation (ROSE) during EUS-FNA would be beneficial; however, many healthcare facilities are unable to introduce this technique.

In situ proteomic analysis by MALDI imaging identifies ubiquitin and thymosin-β4 as markers of malignant intraductal pancreatic mucinous neoplasms

PurposeIntraductal pancreatic mucinous neoplasms (IPMN) are precancerous cystic lesions. The aim was to investigate the in situ IPMN proteome using MALDI (Matrix-Assisted Laser Desorption/Ionisation) imaging and to characterize biomarkers associated with the grade of dysplasia. Experimental designFrozen human Branch duct -IPMN sections were selected according to dysplasia and proteomic analyses were performed by MALDI imaging to obtain mass spectra distribution. The most discriminating peaks were identified using tissue extraction and nanoLC-ESI-MS/MS. Identified peaks were validated in independent series of IPMN by immunochemistry on surgical specimens (tissue-microarrays (TMA), n = 45) and endoscopic ultrasound fine-needle aspiration (EUS FNA) samples (n = 25). ResultsBD-IPMN samples with low (n = 10) and high (n = 10) grades of dysplasia were analyzed. Differential spectra of proteins were found in the two groups with significantly different intensities (n = 15). The two peaks (intense in high grade IPMN) (m/z 8565 and 4747) were characterized as the monomeric ubiquitin (Mascot score = 319.22) and an acetylated fragment of thymosin-β4 (2–42) (Omssa score = 1.37 E−9). Validation on TMA and EUS FNA samples confirmed that ubiquitin was overexpressed in high grade dysplasia (p = 0.04 and p = 0.0004). Thymosin-β4 expression was confirmed on TMA by immunohistochemistry on high grade IPMN (p = 0.011). ConclusionUbiquitin and thymosin-β4 are overexpressed in IPMN with high grade dysplasia. Positive immunochemical staining on EUS-FNA material is a major argument in support of preventive resection.