Abstract
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.
Highlights
Pancreatic ductal adenocarcinoma (PDAC) is predicted to become the second leading cause of cancer-related mortality with a five year survival rate of only 8% [1,2]
Among 1954 endoscopic ultrasound (EUS) performed between November 2018 and October 2019, 37 patients were enrolled with a mean age of 68.3 years ± 10.9 standard deviation (SD) and with
Twenty-four (64.9%) lesions were located in the head of the pancreas with a mean diameter at EUS of 32.8 ± 10.6 mm, with 51.4% being borderline resectable and 35.1% diagnosed as locally advanced (Table 1)
Summary
Pancreatic ductal adenocarcinoma (PDAC) is predicted to become the second leading cause of cancer-related mortality with a five year survival rate of only 8% [1,2]. There are, data reporting a significantly improved overall survival for PDAC patients receiving matched therapies following molecular profiling of the tumor [6]. Two main molecular subtypes of PDAC have been identified, named classical and basal-like, which are characterized by a different prognosis and response to therapy [7,8,9,10,11]. These subtypes have been identified through RNA sequencing studies performed on PDAC tissue derived mostly from surgical specimens. At diagnosis only 20% of PDAC patients are eligible for surgery [12], whereas the majority presents either with a locally advanced or a metastatic disease and is directly addressed to chemotherapic treatments
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