Basic Study
Copyright ©The Author(s) 2024. Published by Baishideng Publishing Group Inc. All rights reserved.
World J Gastrointest Oncol. Jun 15, 2024; 16(6): 2663-2672
Published online Jun 15, 2024. doi: 10.4251/wjgo.v16.i6.2663
Fine-needle aspiration technique under endoscopic ultrasound guidance: A technical approach for RNA profiling of pancreatic neoplasms
Sabina Sherafedinovna Seyfedinova, Olga Aleksandrovna Freylikhman, Polina Sergeevna Sokolnikova, Konstantin Aleksandrovich Samochernykh, Anna Aleksandrovna Kostareva, Olga Viktorovna Kalinina, Evgeniy Gennadievich Solonitsyn
Sabina Sherafedinovna Seyfedinova, Olga Aleksandrovna Freylikhman, Evgeniy Gennadievich Solonitsyn, Research Laboratory of Digestive Cancer, Almazov Medical Research Centre, Saint-Petersburg 197341, Russia
Polina Sergeevna Sokolnikova, Research Laboratory of Molecular and Cellular Modeling and Gene Therapy, Almazov Medical Research Centre, Saint-Petersburg 197341, Russia
Konstantin Aleksandrovich Samochernykh, Head of Russian Neurosurgical Institute Named after Prof. A. L. Polenova, Russian Neurosurgical Institute Named after Prof. A. L. Polenova, Saint-Petersburg 191014, Russia
Anna Aleksandrovna Kostareva, Head of Institute of Molecular Biology and Genetics, Almazov National Medical Research Centre, Saint-Petersburg 197341, Russia
Olga Viktorovna Kalinina, Research Laboratory of Autoimmune and Autoinflammatory Diseases, Almazov National Medical Research Centre, Saint-Petersburg 197341, Russia
Co-first authors: Sabina Sherafedinovna Seyfedinova and Olga Aleksandrovna Freylikhman.
Author contributions: Freylikhman OA as a specialist in molecular biology conducted technical part of the investigation and descripted methods and results in the article, contributed in the methodology development. Seyfedinova SS and Freylikhman OA are co-first authors of this manuscript. Seyfedinova SS, Freylikhman OA, and Sokolnikova PS contributed to the data curation and investigation; Seyfedinova SS, Freylikhman OA, Samochernykh KA, Kostareva AA, Kalinina OV, and Solonitsyn EG were involved in the methodology of this study; Seyfedinova SS and Freylikhman OA participated in the writing-original draft; Samochernykh KA and Kostareva AA contributed to the supervision of this manuscript; Kostareva AA, Kalinina OV, and Solonitsyn EG were involved in the validation and editing of this article; Kalinina OV and Solonitsyn EG contributed to the conceptualization.
Supported by the Ministry of Science and Higher Education of the Russian Federation, No. 075-15-2022-301.
Institutional review board statement: All samples were obtained for scientific research according to the Helsinki Declaration and approved by the Almazov National Medical Centre ethics committee (Approval No. 2101-23).
Informed consent statement: All study participants, or their legal guardians, provided informed written consent before study enrollment.
Conflict-of-interest statement: All the authors report no relevant conflicts of interest for this article.
Data sharing statement: Technical appendix, statistical code, and dataset available from the corresponding author at seysabina000@gmail.com. No additional data are available.
Open-Access: This article is an open-access article that was selected by an in-house editor and fully peer-reviewed by external reviewers. It is distributed in accordance with the Creative Commons Attribution NonCommercial (CC BY-NC 4.0) license, which permits others to distribute, remix, adapt, build upon this work non-commercially, and license their derivative works on different terms, provided the original work is properly cited and the use is non-commercial. See: https://creativecommons.org/Licenses/by-nc/4.0/
Corresponding author: Sabina Sherafedinovna Seyfedinova, Doctor, Research Scientist, Research Laboratory of Digestive Cancer, Almazov Medical Research Centre, Akkuratova Av. 2, Saint-Petersburg 197341, Russia. seysabina000@gmail.com
Received: December 26, 2023
Revised: February 18, 2024
Accepted: April 7, 2024
Published online: June 15, 2024
Processing time: 171 Days and 19.2 Hours
Abstract
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 °C 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 low-quality 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.

Keywords: Endoscopic ultrasound-guided, fine-needle aspiration, Pancreatic cancer, Pancreatic cysts, RNA extraction, Through-the-needle biopsy, Next-generation sequencing

Core Tip: In this study, the use of RNA from endoscopic ultrasound-guided, fine-needle aspiration (EUS-FNA) samples of cystic and solid pancreatic neoplasms, including samples of low-quality RNA, was demonstrated. The process of sample preparation from EUS-FNA material is detailed along with the necessary criteria for assessing RNA quality for effective molecular biological analysis. Issues regarding the dependence of RNA quality on the type of neoplasm and material are identified. Modification of the library preparation protocol made it possible to obtain libraries for samples with RNA integrity number equivalent values of three and above, meeting the quality criteria for purity and concentration.