Endoscopic Ultrasound-guided Fine-needle Aspiration Specimens Research Articles

Tissue processing of endoscopic ultrasound-guided fine-needle aspiration specimens from solid pancreatic lesions.

Now that tissue cores can be obtained using fine-needle biopsy (FNB) needles, the ways tissues are handled for endoscopic ultrasound-guided fine-needle aspiration (EUS-FNA) are changing. Direct smear, touch smear of core tissues, and centrifugation have been used for cytological examinations, and liquid-based cytology (LBC), which allows immunostaining and genetic tests that use residual samples, is emerging as an alternative. We emphasize that liquid cytology (Cytospin™ cytology and LBC) is still important, because it enables the diagnosis of pancreatic ductal adenocarcinoma (PDAC) when cancerous cells are scarce in specimens. Cell blocks are being replaced by core tissues obtained via FNB needles. Recent reports indicate that rapid on-site evaluation (ROSE) is not necessary when FNB needles are used, and macroscopic on-site evaluation is used to evaluate specimen adequacy. Macroscopic findings of specimens are helpful in the diagnostic workup and for clarifying specimen-handling methods. In addition to the red strings and white cores observed in PDAC, mixed red and white strings, gray tissues, and gelatinous tissues are observed. Gray (necrotic) tissues and gelatinous (mucus) tissues are more suitable than histology for cell block or cytological processing. Tumor cells in neuroendocrine tumors (NETs) are numerous in red strings but cannot be observed macroscopically. ROSE might thus be necessary for lesions that may be NETs. Core tissues can be used for genetic tests, such as those used for KRAS mutations and comprehensive genomic profiling. Cytological materials, including slides and LBC specimens, can also be genetic test materials.

Improving fine needle aspiration to predict the tumor biological aggressiveness in pancreatic neuroendocrine tumors using Ki-67 proliferation index, phosphorylated histone H3 (PHH3), and BCL-2

IntroductionSurgery is the only known cure for sporadic pancreatic neuroendocrine tumors (PNETs). Therefore, the prediction of the PNETs biological aggressiveness evaluated on endoscopic ultrasound-guided fine-needle aspiration (EUS-FNA) has a significant impact on clinical management. The proliferation rate of Ki-67 in PNETs can help to predict the biological aggressiveness of the tumor. In addition, there is a relatively new proliferation marker called phosphorylated histone H3 (PHH3) that can identify and quantify dividing cells in tissue samples, which is a marker highly specific to mitotic figures. Other markers such as BCL-2 also contribute to tumorigenesis and may be involved in the differentiation of neuroendocrine cells. Materials and methodsA retrospective observational study was performed on patients undergoing surveillance for PNETs from January 2010 to May 2021. Data collection included the patients' age, sex, tumor location, tumor size in the surgical specimen, and tumor grade in FNA. The 2019 World Health Organization (WHO) classification guideline was followed to diagnose PNETs, including grade and stage. Immunohistochemical stainings for Ki-67, PHH3 and BCL-2 in PNETs were performed. ResultsAfter excluding cell blocks containing fewer than 100 tumor cells, 44 patients with EUS-FNA and surgical resection specimens were included in this study. There were 19 cases of G1 PNETs, 20 cases of G2 PNETs, and 5 cases of G3 PNETs. The grade assigned based on the Ki-67 index was higher and more sensitive than that based on the mitotic count using H&E slides in some cases of G2 and G3 PNETs. However, there was no significant difference between the mitotic count using PHH3-positive tumor cells and the Ki-67 index to grade PNETs.All grade 1 tumors (19 cases) on surgical resection specimens were correctly graded on FNA (100 % concordance rate). Within the 20 G2 PNETs, 15 cases of grade 2 on surgical resection specimens were graded correctly on FNA based on the Ki-67 index only. Five cases of grade 2 PNETs on surgical resection specimens were graded as grade 1 on FNA when using only the Ki-67 index. Three of five grade 3 tumors on surgical resection specimens were graded as grade 2 on FNA based on the Ki-67 index only. Using only FNA Ki-67 to predict PNET tumor grade, the concordance (accuracy) rate was 81.8 % in total. However, all these eight cases (5 cases of G2 PNETs and 3 cases of G3 PNETs) were graded correctly by using the Ki-67 index plus mitotic rate (using PHH3 IHC stains).Four of 18 (22.2 %) patients with PNETs were positive for BCL-2 stain. In these 4 cases positive for BCL-2 stains, 3 cases were G2 PNETs and one case was G3 PNETs. ConclusionGrade and the proliferative rate in EUS-FNA can be used to predict the tumor grade in surgical resection specimens. However, when using only FNA Ki-67 to predict PNET tumor grade, about 18 % of cases were downgraded by one level. To solve the problem, immunohistochemical staining for BCL-2 and especially PHH3 would be helpful. Our results demonstrated that the mitotic count using PHH3 IHC stains not only improved the accuracy and precision of PNET grading in the surgical resection specimens, but also could reliably be used in routine scoring of mitotic figures of FNA specimens.

A case of pancreatic neuroendocrine tumor with amyloid deposition (insulinoma) predicted by rapid on-site evaluation of pancreatic endoscopic ultrasound-guided fine-needle aspiration specimen

A case of pancreatic neuroendocrine tumor with amyloid deposition (insulinoma) predicted by rapid on-site evaluation of pancreatic endoscopic ultrasound-guided fine-needle aspiration specimen

P53 immunolabeling in EUS-FNA biopsy can predict low resection rate and early recurrence in resectable or borderline resectable pancreatic cancer treated with neoadjuvant therapy.

KRAS, P16, TP53, and SMAD4/DPC4 mutations are common in pancreatic ductal adenocarcinoma (PDAC). The study aimed to evaluate the association between gene mutations in pre-treatment endoscopic ultrasound-guided fine needle aspiration (EUS-FNA) samples and clinical outcomes of patients with PDAC. There were 43 patients with resectable (R) PDAC and 41 patients with borderline resectable (BR) PDAC. CDKN2A/p16, TP53, and SMAD4/DPC4 were evaluated through immunohistochemistry (IHC) of pretreatment EUS-FNA (n=84) and resected specimens (n=71). All patients received neoadjuvant therapy. IHC of EUS-FNA specimens revealed p16 loss in 61 (73%), abnormal p53 in 61 (73%), and Smad4 loss in 38 (45%) patients. Abnormal p53 was associated with a lower resection rate (p=.017). Abnormal p53 and Smad4 loss were associated with recurrence within 6 months post-pancreatectomy (p=.03, p=.03, respectively). Univariate Cox regression analysis was conducted to reveal that abnormal p53 (p=.07), p16 loss and abnormal p53 (p=.04), and Smad4 and p16 loss (p=.03) were associated with poor prognosis. Pre-treatment abnormal labeling of p53 in EUS-FNA specimen was associated with a lower resection rate and an early recurrence in R or BR PDAC cases.

Interobserver Agreement among Cytopathologists in False-Negative Cases by Cytological Diagnosis with Endoscopic Ultrasound-Guided Fine Needle Aspiration in Solid Pancreatic Lesions

Introduction: Early detection and accurate pathological assessment are critical to improving prognosis of pancreatic cancer. EUS has been widely used in diagnosing pancreatic lesions and can obtain histological diagnosis by endoscopic ultrasound-guided fine needle aspiration (EUS-FNA). However, comprehensive assessment of the interobserver agreement (IOA) among cytopathologists evaluating EUS-FNA specimens is still limited. Therefore, this study evaluated IOA among cytopathologists for EUS-FNA specimens of solid pancreatic lesions, especially in false-negative cases of cytological diagnosis and analyzed the factors that influence cytological diagnosis of EUS-FNA so as to improve the diagnostic efficiency of EUS-FNA. Methods: We retrieved EUS-FNA samples of pancreatic solid lesions from 2017 to 2021 and collected their clinical/cytological data. Two cytopathologists independently reviewed these cases using a quoted, novel standardized cytology scoring tool. Ultimately, we calculated IOA among cytopathologists and performed a binary logistic regression analysis to evaluate factors influencing the cytological diagnosis of EUS-FNA. Results: 161 patients were included, and 60 cases with a clinical diagnosis of pancreatic cancer but a cytological diagnosis of benign and atypical constituted the false-negative group. IOAs for cytological diagnosis of overall patients and the false-negative group were in perfect/moderate agreement with Kendall’s W values of 0.896 and 0.462, respectively. The number of diagnostic cells in the scoring tool had the highest level of agreement (κ = 0.721) for overall patients. There was at best moderate agreement on other quantity and quality parameters for both all cases and false-negative group. Logistic regression analysis showed the number of diagnostic cells (OR = 6.110, p < 0.05) and amount of blood (OR = 0.320, p < 0.05) could influence cytological diagnosis. Conclusions: The false-negative rate of our study as high as 37.26% (60/161) is mainly related to strict standards of cytopathologists, and their ability to standardize pancreatic cytology is still improving. Suboptimal agreement among cytopathologists for cytological diagnosis and the number of diagnostic cells may be associated with the occurrence of false-negative diagnosis. Further regression analysis confirmed that the number of diagnostic cells and obscuring blood were important factors in cytological diagnosis. Therefore, refinement of cytological diagnostic criteria, standardization of specimen quality evaluation, and training of cytopathologists may improve the agreement of cytopathologists, thus improving the repeatability of cytological diagnosis and reducing the occurrence of false-negative events.

KRAS Mutation Testing on Endoscopic Ultrasound-Guided Fine-Needle Aspiration Samples Improves the Diagnostic Accuracy of Pancreatic Cancer.

Endoscopic ultrasound-guided fine-needle aspiration (EUS-FNA) cytology was one of the investigations for pancreatic masses. While the specificity approached 100%, its sensitivity remained low because of high rate of indeterminate and false-negative results. Meanwhile, KRAS gene was frequently mutated in up to 90% of pancreatic ductal adenocarcinoma and its precursor lesions. This study aimed to determine whether KRAS mutation analysis could improve the diagnostic sensitivity in EUS-FNA samples for pancreatic adenocarcinoma. The EUS-FNA samples from patients with a pancreatic mass obtained between January 2016 and December 2017 were reviewed retrospectively. The cytology results were classified as malignant, suspicious for malignancy, atypical, negative for malignancy, and nondiagnostic. KRAS mutation testing was performed using polymerase chain reaction followed by Sanger sequencing. A total of 126 EUS-FNA specimens were reviewed. The overall sensitivity and specificity by cytology alone were 29% and 100%, respectively. When KRAS mutation testing was performed in cases with indeterminate and negative cytology, the sensitivity increased to 74.2%, and the specificity remained at 100%. KRAS mutation analysis, especially when performed in cytologically indeterminate cases, improves the diagnostic accuracy for pancreatic ductal adenocarcinoma. This may reduce the need to repeat invasive EUS-FNA for diagnosis.

Diagnostic and Prognostic Values of KRAS Mutations on EUS-FNA Specimens and Circulating Tumor DNA in Patients With Pancreatic Cancer.

INTRODUCTION:The ability of carbohydrate antigen 19-9 (CA19-9) to differentiate pancreatic cancer from other benign pancreatic lesions is unsatisfactory. This study explored the diagnostic value of KRAS gene mutations and plasma circulating tumor DNA (ctDNA) in patients with pancreatic cancer.METHODS:The prospective cohort study comprised 149 consecutive patients with solid pancreatic lesions who underwent endoscopic ultrasound-guided fine-needle aspiration (EUS-FNA). KRAS subtype mutations were analyzed by digital droplet PCR (ddPCR) in EUS-FNA histopathology tissue samples, and blood samples were sent for plasma ctDNA analysis. The final diagnosis was based on surgical resection pathology or follow-up for at least 2 years.RESULTS:Adding KRAS mutation ddPCR increased the sensitivity and accuracy of EUS-FNA from 71.4% to 91.6% (P < 0.001) and 75.8% to 88.6% (P < 0.001), respectively. By comparison, the sensitivities of circulating biomarkers ctDNA and CA19-9 were only 35.2% and 71.2%. The area under the curve of the receiver operating characteristic curve (AUC) of EUS-FNA and KRAS ddPCR combination was >0.90 for distinguishing pancreatic cancer from benign lesions, whereas the AUC of EUS-FNA and CA19-9 combination was 0.83. The median survival time was significantly shorter in patients with G12D KRAS mutations than that in patients with other mutations (180 vs 240 days, P < 0.001).DISCUSSION:FNA tissue sample KRAS mutation analysis in tissues significantly improves the diagnostic accuracy of cyto/histopathological evaluation in EUS-FNA samples. The combination of EUS-FNA and tissue sample KRAS ddPCR provided a more accurate method for pancreatic cancer diagnosis, superior to the combination of EUS-FNA and CA19-9/ctDNA. G12D KRAS mutations in pancreatic cancer were independently associated with poor overall survival.

A New Preoperative Scoring System for Predicting Aggressiveness of Non-Functioning Pancreatic Neuroendocrine Neoplasms.

The management of non-functioning pancreatic neuroendocrine neoplasms (NF-PanNENs) is still controversial. This study aimed to develop a new scoring system for treatment decisions at initial diagnosis based on the identification of the predictive factors for aggressive NF-PanNENs. Seventy-seven patients who had been pathologically diagnosed with NF-PanNENs were enrolled. We retrospectively reviewed 13 variables that could be assessed preoperatively. Univariate and multivariate stepwise logistic regression analyses were performed to identify factors for the aggressiveness of NF-PanNENs, and a scoring system was developed by assigning weighted points proportional to their β regression coefficient. Tumor size > 20 mm on contrast-enhanced computed tomography, tumor non-vascularity, and Ki-67 labeling index ≥5% on endoscopic ultrasound-guided fine-needle aspiration specimens were identified as independent factors for predicting the aggressiveness of NF-PanNENs. The new scoring system, developed using the identified factors, had an excellent discrimination ability, with area under the curve of 0.92 (95% CI, 0.85–0.99), and good calibration (p = 0.72, Hosmer-Lemeshow test). Ten-year overall survival rates in low-risk (0 point), intermediate-risk (1 to 2 points), and high-risk (3 to 4 points) groups were 100%, 90.9%, and 24.3%, respectively. This new scoring system would be useful for treatment decisions and prognostic prediction at initial diagnosis.

Cytological scoring for pancreatic specimens obtained by endoscopic ultrasound-guided fine needle aspiration.

Cytological diagnosis of pancreatic specimens obtained by endoscopic ultrasound-guided fine needle aspiration (EUS-FNA) is often challenging because of the small sample size or well-differentiated adenocarcinoma with weak cytological atypia. Therefore, the sensitivity and specificity of cytological diagnosis for pancreatic cancer should be improved. Hence, we aimed to clarify the utility of cytological scoring to distinguish malignant from benign lesions for cytological diagnosis of pancreatic EUS-FNA specimens. Seven reviewers, including four cytotechnologists and three medical doctors, evaluated 20morphological indices in pancreatic specimens obtained by EUS-FNA (malignant, n=111; benign, n=31). Statistical analyses were performed using Fisher's exact test, logistic regression analysis, the area under the receiver operating characteristic curve, and Youden index. Among the 20 indices, there was a high incidence rate (>40%) of the following 13 indices in malignant cases: irregular structure, hyperchromatic nucleus, irregular cell polarity, unclear cell boundaries, nuclear membrane thickening, anisonucleosis, overlapping, irregular nuclei, high nuclear-cytoplasmic ratio, binding decline, the simultaneous appearance of malignant and benign cells, enlarged nucleoli, and background necrosis. When we diagnosed pancreatic specimens using these 13 cytological indices, the cut-off value of 8/9showed the highest Youden index (0.950) as well as high sensitivity and specificity in distinguishing malignant from benign specimens (98% and 97%, respectively). Thirteen cytological indices showed high sensitivity and specificity in differentiating malignant and benign lesions using pancreatic EUS-FNA samples. All 13 indices were important for diagnosing malignancy in the pancreatic cytology smear of EUS-FNA. Further validation studies are required.

A Pilot Randomized Crossover Trial of Wet Suction and Conventional Techniques of Endoscopic Ultrasound-Guided Fine-Needle Aspiration for Upper Gastrointestinal Subepithelial Lesions.

Methods Twenty-six patients with UGI-SELs indicated for EUS-FNA were randomly assigned to the dry-first arm using the dry technique for the first two passes or the wet-first arm using the wet technique for the first two passes using a cross-over design with a ratio of 1 : 1. The primary endpoint was the cellularity score of the EUS-FNA specimens rated on a 4-point scale (0-3). The secondary endpoints were the factors influencing cellularity in each suction technique. Results The mean cellularity score was 1.65 ± 1.20 for the wet technique and 2.00 ± 0.98 for the dry technique (p = 0.068). Logistic regression analysis showed that higher cellularity may be related to the final diagnosis of gastrointestinal stromal tumors in the dry technique and the SEL location in the upper stomach in the wet technique. Conclusion The wet EUS-FNA technique failed to show a potential for improved cellularity of specimens compared to the dry technique for UGI-SELs.

The Need for Histological Preparation of Endoscopic Ultrasound-guided Fine-needle Aspiration Specimens to Diagnose Rare Pancreatic Etiologies.

The Need for Histological Preparation of Endoscopic Ultrasound-guided Fine-needle Aspiration Specimens to Diagnose Rare Pancreatic Etiologies.

Diagnosis of pancreatic solid pseudopapillary neoplasms using cell‐blocks and immunohistochemical evaluation of endoscopic ultrasound‐guided fine needle aspiration biopsy specimens

Preoperative diagnostic imaging of pancreatic solid pseudopapillary neoplasms (SPNs) is challenging. A few studies have investigated the role of endoscopic ultrasound-guided fine needle aspiration (EUS-FNA) for the diagnosis of SPN. We investigated the diagnostic yield of cell-blocks and immunohistochemistry (IHC) for SPN using EUS-FNA specimens without cytological evaluation. We retrospectively analysed the histopathology records of patients with suspected SPN, who underwent EUS-FNA biopsy between January 1997 and January 2020. Diagnosis based on cell-blocks (haematoxylin-eosin staining with complementary IHC) was compared with the definitive surgical diagnosis. This study included 25 patients (24 were women). Patients' mean age was 33.7years (range 12-78years). The most common symptom was abdominal pain. SPN was an incidental finding in 52% of the patients. The mean lesion size was 4.3cm (range 1.2-11.4cm), and the most common endosonographic features included solid-cystic (56%) or solid (40%) tumours. Final diagnoses included SPNs (n = 23) and non-functioning neuroendocrine tumours (n = 2). The overall accuracy of EUS-FNA was 80%. Tumour cells showed immunopositivity for β-catenin, CD10, CD99 and progesterone receptor (PR) in 93.7%, 87.5%, 83.3% and 66.6% of patients, respectively. No SPN showed immunopositivity for chromogranin A. Intention-to-diagnose analysis showed that the diagnostic accuracy of EUS-FNA for SPNs using cell blocks and complementary IHC without cytological evaluation was fairly good. Evaluation of β-catenin, CD 10, CD99 and PR expression must be included in the IHC panel for diagnostic confirmation of SPNs using EUS-FNA biopsy specimens.

Grading of pancreatic neuroendocrine tumors on endoscopic ultrasound-guided fine-needle aspiration using Ki-67 index and 2017 World Health Organization criteria: An analysis of 32 cases.

Objectives:Biological behavior of pancreatic neuroendocrine tumors (Pan NETs) is difficult to predict on morphology alone. The assessment of proliferation by the Ki-67 proliferation index (PI) is considered to be an important prognostic parameter in these tumors and has been endorsed by the 2017 World Health Organization (WHO) grading system for Pan NETs. Although widely accepted on surgical specimens, there is varied opinion on grading of these tumors on cytology samples. This study aimed at classification and grading of Pan NETs on endoscopic ultrasound-guided fine-needle aspiration (EUS-FNA) using the recent 2017 WHO criteria and assess the reliability of Ki-67 grading by comparing it with histology samples wherever available.Material and Methods:Search of cytopathology lab records over a 3-year period (June 2015–May 2018) revealed 33 cases of pancreatic NETs diagnosed on EUS-FNA specimens. Using the guidelines of 2017 WHO classification and grading of Pan NETs, retrospective grading of these Pan NETs was done. They were graded as Grades 1, 2, and 3 well differentiated Pan NETs and poorly differentiated Grade 3 neoplasms based on Ki-67 PI and cytomorphology. Cytomorphological features were compared across the three grades. The cytological grading was then compared with the histological grading where available.Results:Ki-67 grading on cytology was done in 32 cases (22 on cell block and 10 on smears), of which 19 (59.4%) were Grade 1, 8 (25%) were Grade 2, and 5 (15.6%) were Grade 3 tumors. The most common cytomorphological features observed in Grade 1 tumors were small round uniform cells with granular chromatin and prominent plasmacytoid morphology. As the grade increased, tumor cells showed increased pleomorphism, angulated nuclei, and less frequent plasmacytoid cells. Histopathology (biopsy/resected specimens) was available in 11 of the 32 cases. Comparison of grading on cytology and histology showed concordance in ten of the 11 cases (k value = 0.862).Conclusion:Our data suggest that grading of Pan NETs by assessing Ki-67 PI on cytology samples collected by EUS-FNA shows good agreement with that measured on histology samples.

Endoscopic ultrasound-guided needle-based confocal laser endomicroscopy in gastrointestinal subepithelial lesions: Feasibility study.

Needle-based confocal laser endomicroscopy (nCLE) allows for real-time optical biopsies during endoscopic ultrasound-guided fine-needle aspiration (EUS-FNA). Little is known about nCLE imaging of gastrointestinal subepithelial lesions (GI-SEL); therefore, we determined its feasibility. We carried out EUS, nCLE, and finally FNA in 25 patients with GI-SEL between November 2015 and December 2018. We retrospectively compared nCLE findings with pathological findings of EUS-FNA or surgical specimens. For concordance analysis, two endoscopists independently validated representative nCLE images 5months or more after examinations. Adequate sample acquisition rate of EUS-FNA was 67% per needle pass and 96% per patient. EUS-FNA was diagnostic in 80% (20/25), suspicious in 4% (1/25), and nondiagnostic in 16% (4/25). nCLE image acquisition rate was 100% and its concordance rate with final pathology was 88% (22/25), which was not significantly different from diagnostic and suspicious EUS-FNA. nCLE could differentiate GI stromal tumors (GISTs) from leiomyoma, in that GISTs were characterized by contrast-enhanced densely populated spindle cell tumors with unenhanced rod-shaped nuclei in 93% of 14 patients, whereas leiomyomas were characterized by narrower spindle cell tumors with fewer and smaller unenhanced nuclei in 100% of three patients. In rectal metastasis from lung adenocarcinoma, some pleomorphic dark nests were observed. At concordance analysis between the two endoscopists' validation results, κ value was 0.560 (P<0.001), indicating moderate agreement. There were no adverse events associated with nCLE and EUS-FNA. Needle-based confocal laser endomicroscopy can be safe and useful for on-site detection of abnormalities of GI-SEL (UMIN 000013857).

Cell block processing is optimal for assessing endoscopic ultrasound fine needle aspiration specimens of pancreatic mucinous cysts

AimsThe cell block technique for assessing endoscopic ultrasound-guided fine needle aspiration (EUS-FNA) specimens from pancreatic mucinous cystic lesions (MCLs) was systematically evaluated for the first time, including comparisons with three...

Instant messenger smartphone application for endosonographer/cytopathologist real-time interaction at a distance in EUS-FNA for solid pancreatic lesions.

Background and study aims Telecytopathology (TCP) may allow proper and timely evaluation of endoscopic ultrasound-guided fine-needle aspiration (EUS-FNA) specimens. The aim of this study was to evaluate the feasibility of TC through a multiplatform instant messenger smartphone application to evaluate specimens of EUS-FNA of pancreatic solid lesions. Patients and methods Twenty-three patients (14 male/9 female; median age: 56 yr.; age range: 33 – 86) with a solid pancreatic lesion were included. Exclusion criteria were as follows: age < 18 yr and predominantly cystic lesions. During each EUS-FNA, after each pass, the aspirated material was spread over a glass slide and was stained by the endoscopist. The glass slide was then reviewed on a microscope with a smartphone fitted in, and the most representative fields were captured and sent to the cytopathologist using WhatsApp Messenger. Results In initial evaluation using TCP rapid on-site evaluation (ROSE), adequate cellularity of the glass slide was detected in 16 of 23 patients (69.6 %). An initial diagnosis of malignancy (positive or suspicious) was possible in 14 of 23 patients (60.8 %). Conclusion The current study demonstrated the feasibility of a low-cost, Internet-based, telecytopathology system using WhatsApp Messenger to provide ROSE of EUS-FNA slides in patients with solid pancreatic lesions.

Diagnostic ability of artificial intelligence using deep learning analysis of cyst fluid in differentiating malignant from benign pancreatic cystic lesions

The diagnosis of pancreatic cystic lesions remains challenging. This study aimed to investigate the diagnostic ability of carcinoembryonic antigen (CEA), cytology, and artificial intelligence (AI) by deep learning using cyst fluid in differentiating malignant from benign cystic lesions. We retrospectively reviewed 85 patients who underwent pancreatic cyst fluid analysis of surgical specimens or endoscopic ultrasound-guided fine-needle aspiration specimens. AI using deep learning was used to construct a diagnostic algorithm. CEA, carbohydrate antigen 19-9, carbohydrate antigen 125, amylase in the cyst fluid, sex, cyst location, connection of the pancreatic duct and cyst, type of cyst, and cytology were keyed into the AI algorithm, and the malignant predictive value of the output was calculated. Area under receiver-operating characteristics curves for the diagnostic ability of malignant cystic lesions were 0.719 (CEA), 0.739 (cytology), and 0.966 (AI). In the diagnostic ability of malignant cystic lesions, sensitivity, specificity, and accuracy of AI were 95.7%, 91.9%, and 92.9%, respectively. AI sensitivity was higher than that of CEA (60.9%, p = 0.021) and cytology (47.8%, p = 0.001). AI accuracy was also higher than CEA (71.8%, p < 0.001) and cytology (85.9%, p = 0.210). AI may improve the diagnostic ability in differentiating malignant from benign pancreatic cystic lesions.

A Novel Approach for the Genetic Analysis of Biliary Tract Cancer Specimens Obtained Through Endoscopic Ultrasound-Guided Fine Needle Aspiration Using Targeted Amplicon Sequencing.

OBJECTIVES:Biliary tract cancer (BTC) is an aggressive malignant tumor, and biomarker-based clinical trials for this cancer are currently ongoing. Endoscopic ultrasound–guided fine needle aspiration (EUS-FNA) is a safe procedure and enables pathological diagnoses; however, it is uncertain whether a tiny tumor sample of BTC obtained through EUS-FNA can be analyzed for diverse genetic alterations in the development and tolerance of BTC. Thus, we aimed to verify the feasibility of genetic analyses with EUS-FNA samples of BTC.METHODS:Targeted amplicon sequencing using a cancer gene panel with 50 genes was performed with tissue samples of 21 BTC patients obtained through EUS-FNA with a novel rapid on-site process compared with paired peripheral blood samples.RESULTS:Pathogenic gene alterations were successfully identified in 20 out of 21 patients (95.2%) with EUS-FNA specimens of BTC, which included 19 adenocarcinomas and 2 adenosquamous carcinomas. Eighty single nucleotide variants and 8 indels in 39 genes were identified in total, and 28 pathogenic alterations in 14 genes were identified (average, 1.4 alterations per patient). The most common alterations were TP53, KRAS, and CDKN2A in gallbladder carcinoma; TP53, KRAS, PIK3CA, and BRAF in intrahepatic cholangiocarcinoma; and TP53 and SMAD4 in extrahepatic cholangiocarcinoma. Actionable gene alterations (BRAF, NRAS, PIK3CA, and IDH1) were identified in 7 out of 21 patients.CONCLUSIONS:A novel approach in genetic analysis using targeted amplicon sequencing with BTC specimens obtained through EUS-FNA was feasible and enabled us to identify genomic alterations.

Accuracy of grading pancreatic neuroendocrine neoplasms with Ki-67 index in fine-needle aspiration cellblock material.

The aim of this study was to assess the preoperative tumour grade of pancreatic neuroendocrine neoplasms (panNENs) by determining the Ki-67 index in endoscopic ultrasound-guided fine needle aspiration (EUS-FNA) material and to correlate the preoperative tumour grade with the postoperative tumour grade in surgical specimens. We performed a retrospective review of the institutional pathology database over a 10-year period (2007-2017) to identify all cases of panNENs with corresponding preoperative EUS-FNA cytological material and surgical specimens. Fifteen cases with adequate EUS-FNA material (more than 400 tumour cells on cellblock) were identified. The cytological and histological samples were graded based on the mitotic rate and the Ki-67 index in accordance with the 2017 World Health Organisation grading system for panNENs. The tumour grades determined on EUS-FNA cellblock material were compared with the histological tumour grades. Mean age at diagnosis was 64.8±12.7years (range, 38-85years). The grading scores assigned to the cytological and histological samples were concordant in all 15 (100%) cases. Of those, two (13%) cases were scored as grade 1, nine (60%) cases as grade 2 and four (27%) cases as grade 3 tumours. Our study shows that tumour grade in patients with PanNENs can be reliably determined by assessing the Ki-67 index in EUS-FNA specimens based on the 2017 World Health Organisation classification and grading system.

Abstract 3331: Development of a sensitive method to detect aberrant DNA methylation in cfDNA for pancreas cancer diagnosis

Abstract Pancreatic cancer is a highly lethal malignancy despite recent clinical advances. Since pancreatic cancer is often difficult to diagnose, there is an urgent need to develop noninvasive biomarkers for detection. Epigenetic alterations have been recognized as a common hallmark of many types of human cancers as well as pancreatic cancer. The cell-free DNA (cfDNA) in blood possesses tumor-related genetic alterations as well as epigenetic alterations. Therefore, invention of blood-based DNA methylation biomarkers and their high-sensitive detection system may be a promising diagnostic tool for pancreatic cancer. In order to establish the diagnostic biomarkers, first we performed genome-wide DNA methylation analysis of 38 endoscopic ultrasound-guided fine-needle aspiration specimens of pancreatic cancer using Illumina Infinium HumanMethylation450 BeadChip. We further analyzed the DNA methylation profiles of other types of tumors in the public database TCGA (The Cancer Genome Atlas), and identified five marker genes, which were specifically methylated in pancreatic cancers. Analysis of DNA methylation status of pancreas cancers comparing 10 normal tissues in TCGA data set revealed that 99% of tumors were methylated in at least one of the five markers (sensitivity, 98.9%; specificity, 44%). We next examined the DNA methylation status of these markers in the validation cohort (n=46) by bisulfite pyrosequencing or quantitative methylation-specific PCR and found that 42/46 (91.3%) cases were methylated in at least one marker. Finally, we analyzed methylation status in cfDNA from pancreatic cancer patients. We performed bisulfite sequencing using next-generation sequencer and detected methylation in only 5/13 (38.5%) samples in one of the markers. Since the amount of DNA from cancer tissue is very small, we need to develop more a sensitive technique to detect aberrant DNA methylation to diagnose cancer patients. Now we are developing a new sensitive method to detect DNA methylation in cfDNA. Citation Format: Keiko Shinjo, Keisuke Katsushima, Miho Suzuki, Genta Nagae, Hiroyuki Aburatani, Kenji Yamao, Yutaka Kondo. Development of a sensitive method to detect aberrant DNA methylation in cfDNA for pancreas cancer diagnosis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3331.