Fine-needle aspiration technique under endoscopic ultrasound guidance:A technical approach for RNA profiling of pancreatic neoplasms

BACKGROUND Early diagnosis of pancreatic ductal adenocarcinoma(PDAC)has been a longstanding challenge.The prognosis of patients with PDAC depends on the stage at diagnosis.It is necessary to identify biomarkers for the detection and differentiation of pancreatic tumors and optimize PDAC sample preparation procedures for DNA and RNA analysis.Most molecular studies are done using paraffin-embedded blocks;however,the integrity of DNA and RNA is often compromised in this format.Moreover,RNA isolated from human pancreatic tissue samples is generally of low quality,in part,because of the high concentration of endogenous pancreatic RNAse activity present.AIM To assess the potential of endoscopic ultrasound-guided fine-needle aspiration(EUS-FNA)to obtain specimens from pancreatic neoplasms for subsequent RNA molecular profiling,including next-generation sequencing(NGS).METHODS Thirty-four EUS-FNA samples were included in this study:PDAC(n=15),chronic pancreatitis(n=5),pancreatic cysts(n=14),mucinous cysts(mucinous cystic neoplasia/intraductal papillary mucinous neoplasia)n=7,serous cystic neoplasms n=5,and pseudocysts n=2.Cyst material consisted of cyst fluid and cyst wall samples obtained by through-the-needle biopsy(TTNB).Samples were stored at -80℃ until analysis.RNA purity(A260/230,A260/280 ratios),concentration,and integrity(RIN)were assessed.Real-time polymerase chain reaction was conducted on all samples,and small RNA libraries were prepared from solid mass samples.RESULTS RNA was successfully extracted from 29/34(85%)EUS-FNA samples:100% pancreatic adenocarcinoma samples,100% chronic pancreatitis samples,70% pancreatic fluid cyst samples,and 50%TTNB samples.The relative expression of GAPDH and HPRT were obtained for all successfully extracted RNA samples(n=29)including lowquality RNA specimens.Low concentration and nonoptimal RIN values(no less than 3)of RNA extracted from EUS-FNA samples did not prevent NGS library preparation.The suitability of cyst fluid samples for RNA profiling varied.The quality of RNA extracted from mucinous cyst fluid had a median RIN of 7.7(5.0-8.2),which was compatible with that from solid neoplasms[6.2(0-7.8)],whereas the quality of the RNA extracted from all fluids of serous cystic neoplasms and TTNB samples had a RIN of 0.CONCLUSION The results demonstrate the high potential of EUS-FNA material for RNA profiling of various pancreatic lesions,including low-quality RNA specimens.

Endoscopic ultrasound-guided fine-needle aspiration biopsy-Recent topics and technical tips

Endoscopic ultrasound-guided fine-needle aspiration biopsy (EUS-FNA) is a useful procedure that enables reliable pathological diagnoses of pancreatobiliary diseases, subepithelial lesions, and swollen lymph nodes. In recent years, a pathological diagnosis based on EUS-FNA has made it possible to provide accurate treatment methods not only in these fields, but also in respiratory organs and otorhinolaryngology. This review discusses the latest topics pertaining to EUS-FNA as well as procedural tips.

Endoscopic ultrasound-guided tissue acquisition for the diagnosis of focal liver lesion

In patients with liver tumors,the histopathology examination can assist in diagnosis,staging,prognosis,and therapeutic management strategy.Endoscopic ultrasound(EUS)-guided tissue acquisition using fine needle aspiration(FNA)or more newly fine needle biopsy(FNB)is a well-developed technique in order to evaluate and differentiate the liver masses.The goal of the EUS-FNA or EUS-FNB is to provide an accurate sample for a histopathology examination.Therefore,malignant tumors such as hepatocarcinoma,cholangiocarcinoma and liver metastasis or benign tumors such as liver adenoma,focal hyperplastic nodular tumors and cystic lesions can be accurately diagnosed using EUS-guided tissue acquisition.EUS-FNB using 19 or 22 Ga needle provide longer samples and a higher diagnostic accuracy in patients with liver masses when compared with EUS-FNA.Few data are available on the diagnostic accuracy of EUS-FNB when compared with percutaneously,ultrasound,computer tomography or transjugulary-guided liver biopsies.This review will discuss the EUS-guided tissue acquisition options in patients with liver tumors and its efficacy and safety in providing accurate samples.The results of the last studies comparing EUS-guided liver biopsy with other conventional techniques are presented.The EUS-guided tissue acquisition using FNB can be a suitable technique in suspected liver lesions in order to provide an accurate histopathology diagnosis,especially for those who require endoscopy.

Ultrasound-guided fine-needle aspiration/biopsy-based pancreatic organoids establishment:an alternative model for basic and preclinical research

Pancreatic ductal adenocarcinoma(PDAC),as one of the malignant cancers with the worst prognosis,is becoming the most urgent clinical problem.Due to the lack of early diagnosis and curable therapeutic methods,it is critical to exploit proper models that can capture the overall attributes of the primary tumor.Recently,organoid technology has emerged and flourished as a powerful tool to enable long-termculture of pancreatic tissues,including PDAC.As accumulating studies suggest,organoids can retain morphological,genetic,and behavioral traits,and have tremendous value in predicting the therapeutic response to conventional chemotherapy drugs or newfangled agents.Herein,this review comprehensively summarizes the tissue source including human fetal and adult pancreatic tissue to generate a pancreatic organoid as well as current organoids cultivate system.As PDAC organoids can be established from a small number of samples derived from endoscopic ultrasound-guided fine-needle aspiration/biopsy(EUS-FNA/FNB),we also review the literature to date on EUS-FNA/FNBbased organoid constitution and its implementation in inquiring tumor behavior and evaluating therapeutic responses.By enabling the alignment of basic and clinical research platforms,the application of organoids would open up new avenues for drug discovery and maximally benefit translational medicine in the near future.

Endoscopic ultrasound-guided through-the-needle microforceps biopsy and needle-based confocal laser-endomicroscopy increase detection of potentially malignant pancreatic cystic lesions:A single-center study

BACKGROUND Currently,there is insufficient data about the accuracy in the diagnosing of pancreatic cystic lesions(PCLs),especially with novel endoscopic techniques such as with direct intracystic micro-forceps biopsy(mFB)and needle-based confocal laser-endomicroscopy(nCLE).AIM To compare the accuracy of endoscopic ultrasound(EUS)and associated techniques for the detection of potentially malignant PCLs:EUS-guided fine needle aspiration(EUS-FNA),contrast-enhanced EUS(CE-EUS),EUS-guided fiberoptic probe cystoscopy(cystoscopy),mFB,and nCLE.METHODS This was a single-center,retrospective study.We identified patients who had undergone EUS,with or without additional diagnostic techniques,and had been diagnosed with PCLs.We determined agreement among malignancy after 24-mo follow-up findings with detection of potentially malignant PCLs via the EUSguided techniques and/or EUS-guided biopsy when available(EUS malignancy detection).RESULTS A total of 129 patients were included, with EUS performed alone in 47/129. In 82/129 patients,EUS procedures were performed with additional EUS-FNA (21/82), CE-EUS (20/82), cystoscopy(27/82), mFB (36/82), nCLE (44/82). Agreement between EUS malignancy detection and the 24-mo follow-up findings was higher when associated with additional diagnostic techniques thanEUS alone [62/82 (75.6%) vs 8/47 (17%);OR 4.35, 95%CI: 2.70-7.37;P < 0.001]. The highestmalignancy detection accuracy was reached when nCLE and direct intracystic mFB were bothperformed, with a sensitivity, specificity, positive predictive value, negative predictive value andobserved agreement of 100%, 89.4%, 77.8%, 100% and 92.3%, respectively (P < 0.001 comparedwith EUS-alone).CONCLUSIONThe combined use of EUS-guided mFB and nCLE improves detection of potentially malignantPCLs compared with EUS-alone, EUS-FNA, CE-EUS or cystoscopy.

Overlooked risk for needle tract seeding following endoscopic ultrasound-guided minimally invasive tissue acquisition

Endoscopic ultrasound-guided minimally invasive tissue acquisition can be performed by two approaches as follows: Endoscopic ultrasound-guided fineneedle aspiration(EUS-FNA) and endoscopic ultrasound-guided fine-needle biopsy(EUS-FNB). These have been evolved into leading approaches and widely used for the histological diagnosis of tumors in the gastrointestinal tract and adjacent organs. However, the role of EUS-FNA and EUS-FNB in disease diagnosis and evaluation remains controversial. Although the incidence of surgery-associated complications remains low, the consequences of needle tract seeding can be serious or even life-threatening. Recently, increasing case reports of needle tract seeding are emerging, especially caused by EUS-FNA. This complication needs serious consideration. In the present work, we integrated these case reports and the related literature, and summarized the relevant cases and technical characteristics of needle tract seeding caused by EUS-FNA and EUSFNB. Collectively, our findings provided valuable insights into the prevention and reduction of such serious complication.

Mucosal-incision assisted biopsy for suspected gastric gastrointestinal stromal tumors

To evaluate the diagnostic yield of the procedure, mucosal-incision assisted biopsy (MIAB), for the histological diagnosis of gastric gastrointestinal stromal tumor (GIST), we performed a retrospective review of the 27 patients with suspected gastric GIST who underwent MIAB in our hospitals. Tissue samples obtained by MIAB were sufficient to make a histological diagnosis (diagnostic MIAB) in 23 out of the 27 patients, where the lesions had intraluminal growth patterns. Alternatively, the samples were insufficient (non-diagnosticMIAB) in remaining 4 patients, three of whom had gastric submucosal tumor with extraluminal growth patterns. Although endoscopic ultrasound and fine needle aspiration is the gold standard for obtaining tissue specimens for histological and cytological analysis of suspected gastric GISTs, MIAB can be used as an alternative method for obtaining biopsy specimens of lesions with an intraluminal growth pattern.

Clinical course of suspected small gastrointestinal stromal tumors in the stomach

BACKGROUND Gastric subepithelial lesions are frequently encountered during endoscopic examinations,and the majority of them are small and asymptomatic.Among these lesions,gastrointestinal stromal tumors(GISTs)are the major concern for patients and clinicians owing to their malignant potentials.Although previous guidelines suggested periodic surveillance for such small(≤20 mm)lesions,several patients and clinicians have still requested or prescribed repeated examinations or radical resection,posing extra medical burdens and risks.AIM To describe the clinical course of suspected small gastric GISTs and provide further evidence for surveillance strategy for tumor therapy.METHODS This single-center,retrospective study was conducted at West China Hospital,Sichuan University.Consecutive patients with suspected small gastric GISTs were reviewed from November 2004 to November 2018.GIST was suspected according to endoscopic ultrasonography features:hypoechoic lesions from muscularis propria or muscularis mucosa.Eligible patients with suspected small(≤20 mm)GISTs were included for analysis.Patients’demographic data,lesions’characteristics,and follow-up medical records were collected.RESULTS A total of 383 patients(male/female,121/262;mean age,54 years)with 410 suspected small gastric GISTs(1 lesion in 362 patients,2 lesions in 16,3 lesions in4,and 4 lesions in 1)were included for analysis.The most common location was gastric fundus(56.6%),followed by body(29.0%),cardia(12.2%),and antrum(2.2%).After a median follow-up of 28 mo(interquartile range,16-48;range,3-156),402 lesions(98.0%)showed no changes in size,and size of 8 lesions(2.0%)was increased(mean increment,10 mm).Of the 8 lesions with size increment,endoscopic or surgical resection was performed in 6 patients(5 GISTs and 1 leiomyoma).For other 2 remaining patients,unroofing biopsy or endoscopic ultrasound-guided fine-needle aspiration was carried out(2 GISTs),while no further change in size was noted over a period of 62-64 mo.CONCLUSION The majority of suspected small(≤20 mm)gastric GISTs had no size increment during follow-up.Regular endoscopic follow-up without pathological diagnosis may be highly helpful for such small gastric subepithelial lesions.

Role of the preoperative usefulness of the pathological diagnosis of pancreatic diseases

Pancreatic cancer is the fifth leading cause of cancer death and has the lowest survival rate of any solid cancer.Endoscopic ultrasound-guided fine-needle aspiration biopsy(EUS-FNA) is currently capable of providing a cytopathological diagnosis of pancreatic malignancies with a higher diagnostic power,with a sensitivity and specificity of 85%-89% and 98%-99%,compared to pancreatic juice cytology(PJC),whose sensitivity and specificity are only 33.3%-93% and 83.3%-100%.However,EUS-FNA is not effective in the cases of carcinoma in situ and minimally invasive carcinoma because both are undetectable by endoscopic ultrasonography,although PJC is able to detect them.As for the frequency of complications such as post endoscopic retrograde cholangiopancreatography pancreatitis,EUS-FNA is safer than PJC.To diagnose pancreatic cancer appropriately,it is necessary for us to master both procedures so that we can select the best methods of sampling tissues while considering the patient's safety and condition.