Next-generation sequencing of pancreatic cyst wall specimens obtained using micro-forceps for improving diagnostic accuracy

Abstract

Background and study aims Pancreatic cysts are common incidental findings, with an estimated prevalence of 13% to 15% in imaging done for other reasons. Diagnosis often relies on collection of cyst fluid, but tissue sampling using micro-forceps may allow for a more reliable diagnosis and higher yield of DNA for next-generation sequencing (NGS). The primary aim was to assess the performance of NGS in identifying mucinous cyst. The secondary aims were to assess DNA yield between the cyst fluid and cyst wall tissue, complication rate and performance of conventional investigations.

Patients and methods Twenty-four patients referred for endoscopic ultrasound were recruited. Biopsies were taken using micro-forceps and the AmpliSeq Cancer Hotspot panel was used for NGS, a polymerase chain reaction assay targeting several hotspots within 50 genes, including GNAS, KRAS and VHL.

Results The concentration of DNA extracted from 24 cyst wall samples was significantly higher than in the nine of 24 available matched cyst fluid samples. The sensitivity, specificity, and diagnostic accuracy of NGS for diagnosing mucinous cyst were 93%, 50% and 84%; for standard of care, they were -66.6%, 50% and 63.1%; and for standard of care with NGS, they were 100%, 50%, and 89.4% respectively. Cyst wall biopsy was able to diagnose 19 of 24 cysts (4 high risk, 7 intraductal papillary mucinous neoplasms, 4 cysts of mucinous origin, and 4 benign).

Conclusions NGS data correlate well with histology and may aid in diagnosis and risk stratification of pancreatic cysts. Cyst wall biopsy performs well in diagnosing cysts but was inadequate in five of 24 patients.

Keywords

Pancreatobiliary (ERCP/PTCD) - Tissue diagnosis - Endoscopic ultrasonography - Pancreas - Fine-needle aspiration/biopsy - GI Pathology

Publication History

Received: 22 March 2023

Accepted after revision: 15 June 2023

Accepted Manuscript online:
31 August 2023

Article published online:
12 October 2023

• References

• 1 Vincent A, Herman J, Schulick R. et al. Pancreatic cancer. Lancet 2011; 378: 607-620 DOI: 10.1016/S0140-6736(10)62307-0. (PMID: 21620466)
• 2 Carrato A, Falcone A, Ducreux M. et al. A systematic review of the burden of pancreatic cancer in Europe: real-world impact on survival, quality of life and costs. J Gastrointest Cancer 2015; 46: 201-211 DOI: 10.1007/s12029-015-9724-1. (PMID: 25972062)
• 3 Walter FM, Mills K, Mendonca SC. et al. Symptoms and patient factors associated with diagnostic intervals for pancreatic cancer (SYMPTOM pancreatic study): a prospective cohort study. Lancet Gastroenterol Hepatol 2016; 1: 298-306
• 4 Canto MI, Schulick RD, Kamel IR. et al. 415g: screening for familial pancreatic neoplasia: a prospective, multicenter blinded study of EUS, CT, and secretin-MRCP (The NCI-Spore Lustgarten Foundation Cancer of the Pancreas CAPS 3 Study). Gastrointest Endosc 2010; 71: AB119
• 5 Sheel ARG, Harrison S, Sarantitis I. et al. Identification of cystic lesions by secondary screening of familial pancreatic cancer (FPC) kindreds is not associated with the stratified risk of cancer. Am J Gastroenterol 2019; 114: 155-164
• 6 Laffan TA, Horton KM, Klein AP. et al. Prevalence of unsuspected pancreatic cysts on MDCT. Am J Roentgenol 2008; 191: 802-807 DOI: 10.2214/AJR.07.3340. (PMID: 18716113)
• 7 de Jong K, Nio CY, Mearadji B. et al. Disappointing interobserver agreement among radiologists for a classifying diagnosis of pancreatic cysts using magnetic resonance imaging. Pancreas 2012; 41: 278-282
• 8 Farrell JJ. Prevalence, diagnosis and management of pancreatic cystic neoplasms: current status and future directions. Gut Liver 2015; 9: 571-589 DOI: 10.5009/gnl15063. (PMID: 26343068)
• 9 Grenacher L, Strauss A, Bergmann F. et al. Cyst features and risk of malignancy in intraductal papillary mucinous neoplasms of the pancreas: imaging and pathology. Viszeralmedizin 2015; 31: 31-37 DOI: 10.1159/000375254. (PMID: 26288613)
• 10 Keane MG, Afghani E. A Review of the diagnosis and management of premalignant pancreatic cystic lesions. J Clin Med 2021; 10 DOI: 10.3390/jcm10061284. (PMID: 33808853)
• 11 European Study Group on Cystic Tumours of the Pancreas. European evidence-based guidelines on pancreatic cystic neoplasms. Gut 2018; 67: 789-804
• 12 Singhi AD, McGrath K, Brand RE. et al. Preoperative next-generation sequencing of pancreatic cyst fluid is highly accurate in cyst classification and detection of advanced neoplasia. Gut 2017; 67: 2131-2141 DOI: 10.1136/gutjnl-2016-313586. (PMID: 28970292)
• 13 Jones M, Zheng Z, Wang J. et al. Impact of next-generation sequencing on the clinical diagnosis of pancreatic cysts. Gastrointest Endosc 2016; 83: 140-148 DOI: 10.1016/j.gie.2015.06.047. (PMID: 26253016)
• 14 Rosenbaum MW, Jones M, Dudley JC. et al. Next-generation sequencing adds value to the preoperative diagnosis of pancreatic cysts. Cancer Cytopathol 2017; 125: 41-47 DOI: 10.1002/cncy.21775. (PMID: 27647802)
• 15 Paniccia A, Polanco PM, Boone BA. et al. Prospective, multi-institutional, real-time next-generation sequencing of pancreatic cyst fluid reveals diverse genomic alterations that improve the clinical management of pancreatic cysts. Gastroenterology 2023; 164: 117-133
• 16 Khalid A, McGrath KM, Zahid M. et al. The role of pancreatic cyst fluid molecular analysis in predicting cyst pathology. Clin Gastroenterol Hepatol 2005; 3: 967-973 DOI: 10.1016/s1542-3565(05)00409-x. (PMID: 16234041)
• 17 Zhang ML, Arpin RN, Brugge WR. et al. Moray micro forceps biopsy improves the diagnosis of specific pancreatic cysts. Cancer Cytopathol 2018; 126: 414-420 DOI: 10.1002/cncy.21988. (PMID: 29660844)
• 18 Li H, Durbin R. Fast and accurate short read alignment with Burrows-Wheeler transform. Bioinformatics 2009; 25: 1754-1760 DOI: 10.1093/bioinformatics/btp324. (PMID: 19451168)
• 19 DePristo MA, Banks E, Poplin R. et al. A framework for variation discovery and genotyping using next-generation DNA sequencing data. Nat Genet 2011; 43: 491-498 DOI: 10.1038/ng.806. (PMID: 21478889)
• 20 Benjamin D, Sato T, Cibulskis K. et al. Calling Somatic SNVs and Indels with Mutect2. bioRxiv 2019; DOI: 10.1101/861054.
• 21 The 1000 Genomes Project Consortium. A global reference for human genetic variation. Nature 2015; 526: 68-74
• 22 Karczewski KJ, Francioli LC, Tiao G. et al. The mutational constraint spectrum quantified from variation in 141,456 humans. Nature 2020; 581: 434-443
• 23 Cingolani P, Platts A, Wang le L. et al. A program for annotating and predicting the effects of single nucleotide polymorphisms, SnpEff: SNPs in the genome of Drosophila melanogaster strain w1118; iso-2; iso-3. Fly (Austin) 2012; 6: 80-92 DOI: 10.4161/fly.19695. (PMID: 22728672)
• 24 Tate JG, Bamford S, Jubb HC. et al. COSMIC: the Catalogue Of Somatic Mutations In Cancer. Nucleic Acids Res 2019; 47: D941-D947 DOI: 10.1093/nar/gky1015. (PMID: 30371878)
• 25 Singhi AD, Koay EJ, Chari ST. et al. Early detection of pancreatic cancer: opportunities and challenges. Gastroenterology 2019; 156: 2024-2040 DOI: 10.1053/j.gastro.2019.01.259. (PMID: 30721664)
• 26 Balaban VD, Cazacu IM, Pinte L. et al. EUS-through-the-needle microbiopsy forceps in pancreatic cystic lesions: A systematic review. Endosc Ultrasound 2021; 10: 19 DOI: 10.4103/eus.eus_23_20. (PMID: 32611848)
• 27 Siddiqui AA, Shahid H, Shah A. et al. High risk of acute pancreatitis after endoscopic ultrasound-guided fine needle aspiration of side branch intraductal papillary mucinous neoplasms. Endosc Ultrasound 2015; 4: 109 DOI: 10.4103/2303-9027.156728. (PMID: 26020044)
• 28 Al-Haddad M, Wallace MB, Brugge W. et al. Fine-needle aspiration of pancreatic cystic lesions: a randomized study with long-term follow-up comparing standard and flexible needles. Endoscopy 2021; 53: 1132-1140
• 29 Singhi AD, Nikiforova MN, Fasanella KE. et al. Preoperative GNAS and KRAS testing in the diagnosis of pancreatic mucinous cysts. Clin Cancer Res 2014; 20: 4381-4389 DOI: 10.1158/1078-0432.CCR-14-0513. (PMID: 24938521)
• 30 Faias S, Duarte M, Albuquerque C. et al. Clinical Impact of KRAS and GNAS analysis added to CEA and Cytology in Pancreatic cystic fluid obtained by EUS-FNA. Dig Dis Sci 2018; 63: 2351-2361
• 31 Tan MC, Basturk O, Brannon AR. et al. GNAS and KRAS mutations define separate progression pathways in intraductal papillary mucinous neoplasm-associated carcinoma. J Am Coll Surg 2015; 220: 845-854 DOI: 10.1016/j.jamcollsurg.2014.11.029. (PMID: 25840541)
• 32 Shoucair S, Habib JR, Pu N. et al. Comprehensive analysis of somatic mutations in driver genes of resected pancreatic ductal adenocarcinoma reveals KRAS G12D and mutant TP53 combination as an independent predictor of clinical outcome. Ann Surg Oncol 2022; 29: 2720-2731 DOI: 10.1245/s10434-021-11081-z. (PMID: 34792696)
• 33 Khalid A, Zahid M, Finkelstein SD. et al. Pancreatic cyst fluid DNA analysis in evaluating pancreatic cysts: a report of the PANDA study. Gastrointest Endosc 2009; 69: 1095-1102 DOI: 10.1016/j.gie.2008.07.033. (PMID: 19152896)
• 34 Kuboki Y, Fischer CG, Beleva Guthrie V. et al. Single-cell sequencing defines genetic heterogeneity in pancreatic cancer precursor lesions. J Pathol 2019; 247: 347-356
• 35 Rift CV, Melchior LC, Kovacevic B. et al. Targeted next-generation sequencing of EUS-guided through-the-needle-biopsy sampling from pancreatic cystic lesions. Gastrointest Endosc 2022; 97: 50-58.e4