Preoperative next-generation sequencing of pancreatic cyst fluid is highly accurate in cyst classification and detection of advanced neoplasia

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Abstract

Objective

DNA-based testing of pancreatic cyst fluid (PCF) is a useful adjunct to the evaluation of pancreatic cysts (PCs). Mutations in KRAS/ GNAS are highly specific for intraductal papillary mucinous neoplasms (IPMNs) and mucinous cystic neoplasms (MCNs), while TP53/ PIK3CA/ PTEN alterations are associated with advanced neoplasia. A prospective study was performed to evaluate preoperative PCF DNA testing.

Design

Over 43-months, 626 PCF specimens from 595 patients were obtained by endoscopic ultrasound (EUS)-fine needle aspiration and assessed by targeted next-generation sequencing (NGS). Molecular results were correlated with EUS findings, ancillary studies and follow-up. A separate cohort of 159 PCF specimens was also evaluated for KRAS/ GNAS mutations by Sanger sequencing.

Results

KRAS/GNAS mutations were identified in 308 (49%) PCs, while alterations in TP53/PIK3CA/PTEN were present in 35 (6%) cases. Based on 102 (17%) patients with surgical follow-up, KRAS/GNAS mutations were detected in 56 (100%) IPMNs and 3 (30%) MCNs, and associated with 89% sensitivity and 100% specificity for a mucinous PC. In comparison, KRAS/GNAS mutations by Sanger sequencing had a 65% sensitivity and 100% specificity. By NGS, the combination of KRAS/GNAS mutations and alterations in TP53/PIK3CA/PTEN had an 89% sensitivity and 100% specificity for advanced neoplasia. Ductal dilatation, a mural nodule and malignant cytopathology had lower sensitivities (42%, 32% and 32%, respectively) and specificities (74%, 94% and 98%, respectively).

Conclusions

In contrast to Sanger sequencing, preoperative NGS of PCF for KRAS/ GNAS mutations is highly sensitive for IPMNs and specific for mucinous PCs. In addition, the combination of TP53/ PIK3CA/ PTEN alterations is a useful preoperative marker for advanced neoplasia.

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Most cited references 12

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Comparison of Sanger sequencing, pyrosequencing, and melting curve analysis for the detection of KRAS mutations: diagnostic and clinical implications.

Athanasios Tsiatis, Alexis Norris-Kirby, Roy G. Rich … (2010)

Mutations in codons 12 and 13 of the KRAS oncogene are relatively common in colorectal and lung adenocarcinomas. Recent data indicate that these mutations result in resistance to anti-epidermal growth factor receptor therapy. Therefore, we assessed Sanger sequencing, pyrosequencing, and melting curve analysis for the detection of KRAS codon 12/13 mutations in formalin-fixed paraffin-embedded samples, including 58 primary and 42 metastatic colorectal adenocarcinomas, 63 primary and 17 metastatic lung adenocarcinomas, and 20 normal colon samples. Of 180 tumor samples, 62.2% were KRAS mutant positive, and 37.8% were negative. Melting curve analysis yielded no false positive or false negative results, but had 10% equivocal calls. Melting curve analysis also resulted in 4 cases with melting curves inconsistent with either wild-type or codon 12/13 mutations. These patterns were generated from samples with double mutants in codons 12/13 and with mutations outside of codons 12/13. Pyrosequencing yielded no false positive or false negative results as well. However, two samples from one patient yielded a pyrogram that was flagged as abnormal, but the mutation subtype could not be determined. Finally, using an electronic cutoff of 10%, Sanger sequencing showed 11.1% false positives and 6.1% false negatives. In our hands, the limit of detection for Sanger sequencing, pyrosequencing, and melting curve analysis was approximately 15 to 20%, 5%, and 10% mutant alleles, respectively.

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Pancreatic cyst fluid DNA analysis in evaluating pancreatic cysts: a report of the PANDA study.

Asif Khalid, Maliha Zahid, Sydney Finkelstein … (2009)

The role of pancreatic cyst fluid DNA analysis in evaluating pancreatic cysts remains unclear. Our purpose was to evaluate the utility of a detailed DNA analysis of pancreatic cyst fluid to diagnose mucinous and malignant cysts. Prospective, multicenter study. Patients with pancreatic cysts presenting for EUS evaluation. EUS-guided pancreatic cyst aspirates cytology evaluation, carcinoembryonic antigen (CEA) level determination, and a detailed DNA analysis; incorporating DNA quantification, k-ras mutation and multiple allelic loss analysis, mutational amplitude, and sequence determination. Cyst fluid analysis compared with surgical pathologic or malignant cytologic examination. The study cohort consisted of 113 patients with 40 malignant, 48 premalignant, and 25 benign cysts. Cyst fluid k-ras mutation was helpful in the diagnosis of mucinous cysts (odds ratio 20.9, specificity 96%), whereas receiver-operator characteristic curve analysis indicated optimal cutoff points for allelic loss amplitude (area under the curve [AUC] 0.79; optimal value > 65%) and CEA (AUC 0.74; optimal value >148 ng/mL). Components of DNA analysis detecting malignant cysts included allelic loss amplitude over 82% (AUC 0.9) and high DNA amount (optical density ratio >10, AUC 0.79). The criteria of a high amplitude k-ras mutation followed by allelic loss showed maximum specificity (96%) for malignancy. All malignant cysts with negative cytologic evaluation (10/40) could be diagnosed as malignant by using DNA analysis. Limited follow-up, selection bias. Elevated amounts of pancreatic cyst fluid DNA, high-amplitude mutations, and specific mutation acquisition sequences are indicators of malignancy. The presence of a k-ras mutation is also indicative of a mucinous cyst. DNA analysis should be considered when cyst cytologic examination is negative for malignancy.

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Cystic precursors to invasive pancreatic cancer.

Anirban Maitra, R Hruban, Hanno Matthaei … (2011)

Improvements in the sensitivity and quality of cross-sectional imaging have led to increasing numbers of patients being diagnosed with cystic lesions of the pancreas. In parallel, clinical, radiological, pathological and molecular studies have improved the systems for classifying these cysts. Patients with asymptomatic serous cystic neoplasms can be managed conservatively with regular monitoring; however, the clinical management of patients with intraductal papillary mucinous neoplasms and mucinous cystic neoplasms is far more challenging, as it is difficult to determine whether these lesions will progress to malignancy. Fortunately, prospective studies have helped to establish that proposed clinical and radiological criteria (the Sendai guidelines) can be used to guide the care of patients with cystic lesions of the pancreas. Despite this progress in imaging and clinical guidelines, sensitive and specific tests have not yet been developed that can reliably predict the histology and biological properties of a cystic lesion. Such biomarkers are urgently needed, as noninvasive precursors of pancreatic cancer are curable, while the vast majority of invasive pancreatic adenocarcinomas are not.

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Author and article information

Journal

Journal ID (nlm-ta): Gut

Journal ID (iso-abbrev): Gut

Journal ID (hwp): gutjnl

Journal ID (publisher-id): gut

Title: Gut

Publisher: BMJ Publishing Group (BMA House, Tavistock Square, London, WC1H 9JR )

ISSN (Print): 0017-5749

ISSN (Electronic): 1468-3288

Publication date (Print): December 2018

Publication date (Electronic): 28 September 2017

Volume: 67

Issue: 12

Pages: 2131-2141

Affiliations

[1 ] departmentDepartment of Pathology , University of Pittsburgh Medical Center Health System , Pittsburgh, Pennsylvania, USA

[2 ] departmentDepartment of Medicine , University of Pittsburgh , Pittsburgh, Pennsylvania, USA

[3 ] departmentDepartment of Surgery , University of Pittsburgh Medical Center , Pittsburgh, Pennsylvania, USA

[4 ] departmentDepartment of Radiology , University of Pittsburgh , Pittsburgh, Pennsylvania, USA

Author notes

[Correspondence to ] Dr Aatur D Singhi, Department of Pathology, University of Pittsburgh Medical Center Health System, Pittsburgh, Pennsylvania, PA 15213, USA; singhiad@ 123456upmc.edu

Article

Publisher ID: gutjnl-2016-313586

DOI: 10.1136/gutjnl-2016-313586

PMC ID: 6241612

PubMed ID: 28970292

SO-VID: b3b06f98-f685-4ce4-b2a9-fca54db015e1

License:

This is an open access article distributed in accordance with the Creative Commons Attribution Non Commercial (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: http://creativecommons.org/licenses/by-nc/4.0/

History

Date received : 13 December 2016

Date revision received : 29 August 2017

Date accepted : 14 September 2017

Funding

Funded by: FundRef http://dx.doi.org/10.13039/100001784, Pancreatic Cancer Action Network;

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ScienceOpen disciplines: Gastroenterology & Hepatology

Keywords: pancreatic cancer,pancreatic pathology,pancreatic epidemiology,pancreato-biliary disorders

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ScienceOpen disciplines: Gastroenterology & Hepatology

Keywords: pancreatic cancer, pancreatic pathology, pancreatic epidemiology, pancreato-biliary disorders

Preoperative next-generation sequencing of pancreatic cyst fluid is highly accurate in cyst classification and detection of advanced neoplasia

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Karger: Gastroenterology

Most cited references 12

Comparison of Sanger sequencing, pyrosequencing, and melting curve analysis for the detection of KRAS mutations: diagnostic and clinical implications.

Pancreatic cyst fluid DNA analysis in evaluating pancreatic cysts: a report of the PANDA study.

Cystic precursors to invasive pancreatic cancer.

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