RNA Extraction from Endoscopic Ultrasound-Acquired Tissue of Pancreatic Cancer Is Feasible and Allows Investigation of Molecular Features

Published on Nov 30, 2020in Cells4.366
· DOI :10.3390/CELLS9122561
Livia Archibugi13
Estimated H-index: 13
Veronica Ruta4
Estimated H-index: 4
(UCSC: Catholic University of the Sacred Heart)
+ 10 AuthorsGabriele Capurso56
Estimated H-index: 56
Transcriptome analyses allow the distinguishing of pancreatic ductal adenocarcinoma (PDAC) subtypes, exhibiting different prognoses and chemotherapy responses. However, RNA extraction from pancreatic tissue is cumbersome and has been performed mainly from surgical samples, which are representative of < 20% of cases. The majority of PDAC patients undergo endoscopic ultrasound (EUS)-guided tissue acquisition (EUS-TA), but RNA has been rarely extracted from EUS-TA with scanty results. Herein, we aimed to determine the best conditions for RNA extraction and analysis from PDAC EUS-TA samples in order to carry out molecular analyses. PDAC cases underwent diagnostic EUS-TA, with needles being a 25G fine needle aspiration (FNA) in all patients and then either a 20G lateral core-trap fine needle biopsy (FNB) or a 25G Franseen FNB; the conservation methods were either snap freezing, RNALater or Trizol. RNA concentration and quality (RNA integrity index; RIN) were analyzed and a panel of genes was investigated for tissue contamination and markers of molecular subtype and aggressivity through qRT-PCR. Seventy-four samples from 37 patients were collected. The median RNA concentration was significantly higher in Trizol samples (10.33 ng/uL) compared with snap frozen (0.64 ng/uL; p < 0.0001) and RNALater (0.19 ng/uL; p < 0.0001). The RIN was similar between Trizol (5.15) and snap frozen samples (5.85), while for both methods it was higher compared with RNALater (2.7). Among the needles, no substantial difference was seen in terms of RNA concentration and quality. qRT-PCR analyses revealed that samples from all needles were suitable for the detection of PDAC subtype markers (GATA6 and ZEB1) and splice variants associated with mutational status (GAP17) as well as for the detection of contaminating tissue around PDAC cells. This is the first study that specifically investigates the best methodology for RNA extraction from EUS-TA. A higher amount of good quality RNA is obtainable with conservation in Trizol with a clear superiority of neither FNA nor FNB needles. RNA samples from EUS-TA are suitable for transcriptome analysis including the investigation of molecular subtype and splice variants expression.
ABSTRACT Background & Aims Given the lack of procedure standardization, findings vary from analyses of pancreatic tissues collected by endoscopic ultrasound-guided fine-needle biopsy. It is not clear which needle and technique yield best specimen for analysis. We compared the specimen quality and accuracy of diagnoses made from samples collected by fine-needle biopsy needles using different collection techniques. Methods Patients found to have pancreatic masses during imaging (n=129) were random...
6 CitationsSource
#1Silvia CarraraH-Index: 20
#1Silvia CarraraH-Index: 12
Last. Alessandro Repici (Humanitas University)H-Index: 71
view all 14 authors...
Abstract Background and Aims EUS-guided biopsy is the method of choice for obtaining pancreatic tissue. Next-generation sequencing (NGS) has been applied to EUS-guided biopsies and may classify patients based on specific molecular profiles. Our study aimed to compare side-by-side the diagnostic yield achievable by genetic identification of somatic mutations detected with NGS versus histological and cytological typing in locally advanced pancreatic carcinoma (LAPC) in samples acquired under EUS g...
4 CitationsSource
#1Valentina Panzeri (UCSC: Catholic University of the Sacred Heart)H-Index: 5
#2Isabella ManniH-Index: 20
Last. Claudio Sette (UCSC: Catholic University of the Sacred Heart)H-Index: 59
view all 12 authors...
Pancreatic ductal adenocarcinoma (PDAC) is a highly aggressive cancer. Most patients present with advanced disease at diagnosis, which only permits palliative chemotherapeutic treatments. RNA dysregulation is a hallmark of most human cancers, including PDAC. To test the impact of RNA processing dysregulation on PDAC pathology, we performed a bioinformatics analysis to identify RNA-binding proteins (RBPs) associated with prognosis. Among the 12 RBPs associated with progression-free survival, we f...
4 CitationsSource
#1Kazunaga Ishigaki (UTokyo: University of Tokyo)H-Index: 10
#2Yousuke Nakai (UTokyo: University of Tokyo)H-Index: 51
Last. Kazuhiko Koike (UTokyo: University of Tokyo)H-Index: 5
view all 17 authors...
Background/Aims Recently, a three-plane symmetric needle with Franseen geometry was developed for endoscopic ultrasound-guided fine needle biopsy (EUS-FNB). In this retrospective study, tissue acquisition per pass was compared between 22-gauge Franseen FNB and standard fine needle aspiration (FNA) needles in patients with solid pancreatic lesions. Methods Consecutive patients who underwent EUS-FNA or EUS-FNB for solid pancreatic lesions between October 2014 and March 2018 were retrospectively st...
6 CitationsSource
#1Shilpa PatilH-Index: 3
#2Benjamin SteuberH-Index: 2
Last. Elisabeth Hessmann (GAU: University of Göttingen)H-Index: 13
view all 21 authors...
Recent studies have thoroughly described genome-wide expression patterns defining molecular subtypes of pancreatic ductal adenocarcinoma (PDAC), with different prognostic and predictive implications. Although the reversible nature of key regulatory transcription circuits defining the two extreme PDAC subtype lineages “classical” and “basal-like” suggests that subtype states are not permanently encoded but underlie a certain degree of plasticity, pharmacologically actionable drivers of PDAC subty...
4 CitationsSource
#1Maria Victoria Recouvreux (DI: Discovery Institute)H-Index: 2
#2Matthew R. Moldenhauer (DI: Discovery Institute)H-Index: 2
Last. Cosimo Commisso (DI: Discovery Institute)H-Index: 6
view all 9 authors...
Tumor cells rely on glutamine to fulfill their metabolic demands and sustain proliferation. The elevated consumption of glutamine can lead to intratumoral nutrient depletion, causing metabolic stress that has the potential to impact tumor progression. Here, we show that nutrient stress caused by glutamine deprivation leads to the induction of epithelial-mesenchymal transition (EMT) in pancreatic ductal adenocarcinoma (PDAC) cells. Mechanistically, we demonstrate that glutamine deficiency regulat...
12 CitationsSource
#1Natalia Juiz (AMU: Aix-Marseille University)H-Index: 4
#2Abdessamad El-Kaoutari (AMU: Aix-Marseille University)H-Index: 6
Last. Nelson Dusetti (AMU: Aix-Marseille University)H-Index: 40
view all 8 authors...
Pancreatic ductal adenocarcinoma (PDAC) is composed of stromal, immune, and cancerous epithelial cells. Transcriptomic analysis of the epithelial compartment allows classification into different phenotypic subtypes as classical and basal-like. However, little is known about the intra-tumor heterogeneity particularly in the epithelial compartment. Growing evidences suggest that this phenotypic segregation is not so precise and different cancerous cell types may coexist in a single tumor. To test ...
19 CitationsSource
#1Lei Zhou (PRC: China Medical University (PRC))H-Index: 2
#2Weiwei Sheng (PRC: China Medical University (PRC))H-Index: 4
Last. Ming Dong (PRC: China Medical University (PRC))H-Index: 23
view all 10 authors...
Our previous studies found overexpression of Musashi2 (MSI2) conduced to the progression and chemoresistance of pancreatic cancer (PC) by negative regulation of Numb and wild type p53 (wtp53). Now, we further investigated the novel signalling involved with MSI2 in PC. We identified inositol-3-phosphate synthase 1 (ISYNA1) as a novel tumour suppressor regulated by MSI2. High MSI2 and low ISYNA1 expression were prevalently observed in 91 PC tissues. ISYNA1 expression was negatively correlated with...
3 CitationsSource
#2Alex Penson (MSK: Memorial Sloan Kettering Cancer Center)H-Index: 4
Last. Steven D. LeachH-Index: 66
view all 44 authors...
Summary Pancreatic ductal adenocarcinoma (PDAC) is driven by co-existing mutations in KRAS and TP53. However, how these mutations collaborate to promote this cancer is unknown. Here, we uncover sequence-specific changes in RNA splicing enforced by mutant p53 which enhance KRAS activity. Mutant p53 increases expression of splicing regulator hnRNPK to promote inclusion of cytosine-rich exons within GTPase-activating proteins (GAPs), negative regulators of RAS family members. Mutant p53-enforced GA...
20 CitationsSource
#1Davendra Sohal (UC: University of Cincinnati)H-Index: 26
#2Erin B. Kennedy (American Society of Clinical Oncology)H-Index: 18
Last. Daniel A. Laheru (Johns Hopkins University)H-Index: 63
view all 19 authors...
PURPOSEThe aim of this work was to provide an update to the ASCO guideline on metastatic pancreatic cancer pertaining to recommendations for therapy options after first-line treatment.METHODSASCO c...
27 CitationsSource
Cited By2
#1Marc HilmiH-Index: 4
#2Jérôme Cros (University of Paris)H-Index: 32
Last. Rémy NicolleH-Index: 12
view all 11 authors...
BACKGROUND Few patients with pancreatic adenocarcinoma (PAC) are eligible for surgery. Patients with early relapse have a poor prognosis and might be better candidates for a medical approach. Clinical and pathological parameters only partially predict recurrence and are only obtained after surgery. PAC subtypes based on gene expression were proposed, and we assessed if they could predict the risk and type of recurrence independently of clinicopathological parameters. METHODS Patients with curati...
1 CitationsSource
#1Eisuke Iwasaki (Keio: Keio University)H-Index: 18
#2Seiichiro Fukuhara (Keio: Keio University)H-Index: 11
Last. Marin Noda (Keio: Keio University)
view all 12 authors...
Pancreatic cancer is the most lethal solid malignancy, and the number of patients with pancreatic cancer is increasing. Systemic chemotherapies are often ineffective for such patients, and there is an urgent need for personalized medicine. Unlike other types of cancer, personalized treatments for pancreatic cancer are still in development. Consequently, pancreatic cancer is less sensitive to anticancer drugs and is often refractory to common treatments. Therefore, advances in personalized medici...