Prospective, Multi-Institutional, Real-Time Next-Generation Sequencing of Pancreatic Cyst Fluid Reveals Diverse Genomic Alterations That Improve the Clinical Management of Pancreatic Cysts

Abstract

Background & aims: Next-generation sequencing (NGS) of pancreatic cyst fluid is a useful adjunct in the assessment of patients with pancreatic cyst. However, previous studies have been retrospective or single institutional experiences. The aim of this study was to prospectively evaluate NGS on a multi-institutional cohort of patients with pancreatic cyst in real time.

Methods: The performance of a 22-gene NGS panel (PancreaSeq) was first retrospectively confirmed and then within a 2-year timeframe, PancreaSeq testing was prospectively used to evaluate endoscopic ultrasound-guided fine-needle aspiration pancreatic cyst fluid from 31 institutions. PancreaSeq results were correlated with endoscopic ultrasound findings, ancillary studies, current pancreatic cyst guidelines, follow-up, and expanded testing (Oncomine) of postoperative specimens.

Results: Among 1933 PCs prospectively tested, 1887 (98%) specimens from 1832 patients were satisfactory for PancreaSeq testing. Follow-up was available for 1216 (66%) patients (median, 23 months). Based on 251 (21%) patients with surgical pathology, mitogen-activated protein kinase/GNAS mutations had 90% sensitivity and 100% specificity for a mucinous cyst (positive predictive value [PPV], 100%; negative predictive value [NPV], 77%). On exclusion of low-level variants, the combination of mitogen-activated protein kinase/GNAS and TP53/SMAD4/CTNNB1/mammalian target of rapamycin alterations had 88% sensitivity and 98% specificity for advanced neoplasia (PPV, 97%; NPV, 93%). Inclusion of cytopathologic evaluation to PancreaSeq testing improved the sensitivity to 93% and maintained a high specificity of 95% (PPV, 92%; NPV, 95%). In comparison, other modalities and current pancreatic cyst guidelines, such as the American Gastroenterology Association and International Association of Pancreatology/Fukuoka guidelines, show inferior diagnostic performance. The sensitivities and specificities of VHL and MEN1/loss of heterozygosity alterations were 71% and 100% for serous cystadenomas (PPV, 100%; NPV, 98%), and 68% and 98% for pancreatic neuroendocrine tumors (PPV, 85%; NPV, 95%), respectively. On follow-up, serous cystadenomas with TP53/TERT mutations exhibited interval growth, whereas pancreatic neuroendocrine tumors with loss of heterozygosity of ≥3 genes tended to have distant metastasis. None of the 965 patients who did not undergo surgery developed malignancy. Postoperative Oncomine testing identified mucinous cysts with BRAF fusions and ERBB2 amplification, and advanced neoplasia with CDKN2A alterations.

Conclusions: PancreaSeq was not only sensitive and specific for various pancreatic cyst types and advanced neoplasia arising from mucinous cysts, but also reveals the diversity of genomic alterations seen in pancreatic cysts and their clinical significance.

Keywords: Diagnosis; Early Detection; Pancreas; Pancreatic Cancer; Pancreatic Neoplasm.

Figure 1.

(A) A summary of the study design to include details of individual patient cohorts used for PancreaSeq testing (tan) and individual analyses performed (blue). (B) Correlative genomic findings based on retrospective PancreaSeq testing of 97 preoperative pancreatic cyst fluid specimens from 63 mucinous cysts and 34 nonmucinous cysts. Among the 63 mucinous cysts, 22 cysts also harbored high-grade dysplasia and/or invasive adenocarcinoma (advanced neoplasia). Genomic alterations in KRAS, GNAS, and/or BRAF were 100% specific for mucinous cysts, whereas alterations in TP53, SMAD4, and/or the mTOR genes were preferentially seen in mucinous cysts with advanced neoplasia. Similarly, genomic alterations in MEN1 and VHL were highly specific for cystic PanNETs and SCAs, respectively. The mTOR genes include PIK3CA and PTEN. HGD, high-grade dysplasia; LGD, low-grade dysplasia.

Figure 2.

(A) An area-proportional Venn diagram demonstrates the distribution of KRAS, GNAS, BRAF, NRAS, and HRAS mutations identified through prospective PancreaSeq testing of 1887 pancreatic cysts. In addition to KRAS and GNAS, BRAF alterations were often identified in EUS-FNA obtained pancreatic cyst fluid specimens and frequently co-occurred with GNAS mutations. (B) Most BRAF alterations found in pancreatic cysts were non-V600E mutations and were predominantly categorized as class II and class III BRAF mutations (n = 83, 91 %). (C) Based on correlative imaging and pathologic studies, BRAF-mutant pancreatic cysts (white arrowhead) were commonly found to communicate with the main pancreatic duct, and (D) on gross pathology, exhibited abundant, thick mucin (white arrowheads). (E and F) Microscopically, BRAF-mutant cysts corresponded to an intraductal papillary mucinous neoplasm with prominent papillary fronds and often lined by both gastric and intestinal epithelium. (E) Hematoxylin and eosin stain, magnification 40×. (F) Hematoxylin and eosin stain, magnification 200×.

Figure 3.

A summary of clinical presentation, imaging findings, pathologic features, preoperative PancreaSeq testing, and postoperative PancreaSeq/Oncomine results for 251 patients with pancreatic cyst with diagnostic surgical pathology. Preoperative genomic alterations involving KRAS, GNAS, and/or BRAF corresponded to the presence of a mucinous cyst, whereas additional alterations in TP53, SMAD4, CTNNB1, and/or the mTOR genes were preferentially found in mucinous cysts with advanced neoplasia. Other key findings were the preoperative detection of LOH for multiple genes that correlated with the presence of a cystic PanNET, and the identification of TP53 and TERT promoter mutations in large SCAs. Postoperative PancreaSeq/Oncomine testing revealed the presence of novel BRAF fusion genes and ERBB2 amplification in RAS wild-type IPMNs (Supplementary Figure 3). Moreover, CDKN2A alterations were preferentially found in IPMNs with advanced neoplasia. MAPK genes include KRAS, BRAF, FIRAS, ERBB2, and MAPK1, and mTOR genes include PTEN, PIK3CA, and AKT1.

Figure 4.

Representative examples of diagnostic surgical pathology for IPMNs that had preoperative PancreaSeq testing. (A) A branch-duct IPMN that was resected because of the presence of a mural nodule (white arrowhead) detected on preoperative imaging. (B) The mural nodule corresponded to collapsed papillary fronds and (C) microscopically, correlated with low-grade dysplasia. Preoperative PancreaSeq testing detected the presence of KRAS and GNAS mutations, but an absence of TP53, SMAD4, CTNNB1, with mTOR gene alterations. (D) A branch-duct IPMN (white arrowhead) with focal ductal dilation and otherwise no concerning preoperative clinical, imaging, or preoperative pathologic findings. Preoperative PancreaSeq testing identified mutations in KRAS and GNAS, and LOH for PTEN and TP53. (E and F) Diagnostic surgical pathology revealed the presence of high-grade dysplasia. (G) A branch-duct IPMN (white arrowhead) with focal ductal dilatation and otherwise no concerning preoperative clinical, imaging, or preoperative pathologic findings. PancreaSeq testing detected a KRAS mutation and a low-level TP53 mutation. Although the submitting surgical pathology report documented the presence of an IPMN with low-grade dysplasia, a (H) focal area of cytologic atypia was identified and (I) corresponded to aberrant nuclear p53 expression. (J) A 3.0-cm branch-duct IPMN (white arrowhead) with otherwise no concerning preoperative clinical, imaging, or preoperative pathologic findings; however, PancreaSeq testing identified a KRAS mutation and SMAD4 LOH. (K) Although histologically consistent with an IPMN with low-grade dysplasia, (L) diffuse loss of Smad4 expression was seen throughout the IPMN. The mTOR genes include PIK3CA and PTEN. (B) Hematoxylin and eosin stain, magnification 20×. (C) Hematoxylin and eosin stain, magnification 200×. (E) Hematoxylin and eosin stain, magnification 20×. (F) Hematoxylin and eosin stain, magnification 200×. (H) Hematoxylin and eosin stain, magnification 200×. (I) p53 immunolabeling, magnification 200×. (K) Hematoxylin and eosin stain, magnification 200×. (L) SMAD4 immunolabeling, magnification 200×.

Figure 5.

(A) A summary of imaging findings, preoperative PancreaSeq testing, and postoperative clinicopathologic features of 87 PanNET patients. Both solid and cystic PanNETs exhibited similar genomic alterations; however, LOH for multiple genes correlated with several adverse clinicopathologic features, such as lymphovascular invasion, perineural invasion, higher T- and N-stage, distant metastases, loss of ATRX/DAXX expression, and the presence of ALT. (B) A representative example of a 1.5-cm PanNET (white arrowhead) in the pancreatic body that preoperative PancreaSeq testing revealed LOH for 4 genes. (C) Microscopically and immunohistochemically, the PanNET was classified as WHO grade 1. (D) However, within a single regional lymph node, a metastasis was identified (black arrowhead). In addition, the PanNET exhibited loss of ATRX expression and the presence of ALT. (C) Hematoxylin and eosin stain, magnification 400×. (D) Hematoxylin and eosin stain, magnification 200×.