Fine-needle aspiration technique under endoscopic ultrasound guidance: A technical approach for RNA profiling of pancreatic neoplasms
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...
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| Published in: | World journal of gastrointestinal oncology Vol. 16; no. 6; pp. 2663 - 2672 |
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| Main Authors: | , , , , , , |
| Format: | Journal Article |
| Language: | English |
| Published: |
China
Baishideng Publishing Group Inc
15-06-2024
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| Subjects: | |
| Online Access: | Get full text |
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| Summary: | 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.
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).
Thirty-four EUS-FNA samples were included in this study: PDAC (
= 15), chronic pancreatitis (
= 5), pancreatic cysts (
= 14), mucinous cysts (mucinous cystic neoplasia/intraductal papillary mucinous neoplasia)
= 7, serous cystic neoplasms
= 5, and pseudocysts
= 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.
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 (
= 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.
The results demonstrate the high potential of EUS-FNA material for RNA profiling of various pancreatic lesions, including low-quality RNA specimens. |
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| Bibliography: | ObjectType-Article-1 SourceType-Scholarly Journals-1 ObjectType-Feature-2 content type line 23 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 Supported by the Ministry of Science and Higher Education of the Russian Federation, No. 075-15-2022-301. 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. |
| ISSN: | 1948-5204 1948-5204 |
| DOI: | 10.4251/wjgo.v16.i6.2663 |