A Combined DNA/RNA-based Next-Generation Sequencing Platform to Improve the Classification of Pancreatic Cysts and Early Detection of Pancreatic Cancer Arising From Pancreatic Cysts

Abstract

Objective: We report the development and validation of a combined DNA/RNA next-generation sequencing (NGS) platform to improve the evaluation of pancreatic cysts.

Background and aims: Despite a multidisciplinary approach, pancreatic cyst classification, such as a cystic precursor neoplasm, and the detection of high-grade dysplasia and early adenocarcinoma (advanced neoplasia) can be challenging. NGS of preoperative pancreatic cyst fluid improves the clinical evaluation of pancreatic cysts, but the recent identification of novel genomic alterations necessitates the creation of a comprehensive panel and the development of a genomic classifier to integrate the complex molecular results.

Methods: An updated and unique 74-gene DNA/RNA-targeted NGS panel (PancreaSeq Genomic Classifier) was created to evaluate 5 classes of genomic alterations to include gene mutations (e.g., KRAS, GNAS, etc.), gene fusions and gene expression. Further, CEA mRNA ( CEACAM5 ) was integrated into the assay using RT-qPCR. Separate multi-institutional cohorts for training (n=108) and validation (n=77) were tested, and diagnostic performance was compared to clinical, imaging, cytopathologic, and guideline data.

Results: Upon creation of a genomic classifier system, PancreaSeq GC yielded a 95% sensitivity and 100% specificity for a cystic precursor neoplasm, and the sensitivity and specificity for advanced neoplasia were 82% and 100%, respectively. Associated symptoms, cyst size, duct dilatation, a mural nodule, increasing cyst size, and malignant cytopathology had lower sensitivities (41-59%) and lower specificities (56-96%) for advanced neoplasia. This test also increased the sensitivity of current pancreatic cyst guidelines (IAP/Fukuoka and AGA) by >10% and maintained their inherent specificity.

Conclusions: PancreaSeq GC was not only accurate in predicting pancreatic cyst type and advanced neoplasia but also improved the sensitivity of current pancreatic cyst guidelines.

FIGURE 1

A pancreatic cyst that was evaluated by PancreaSeq GC. A and B, An incidentally identified 1.2 cm cyst (white arrowhead, pancreatic cyst) that communicated with the pancreatic duct and clinically suspicious for a branch-duct IPMN without concerning imaging features. C, Cytopathology of EUS-FNA pancreatic cyst fluid detected atypical ductal cells in a background of debris. Upon surgical resection, D, the macroscopic (yellow arrowhead, pancreatic cyst) and E, microscopic findings were consistent with an IPMN involving the side-branch. F, In addition, high-grade dysplasia was identified within the IPMN; however, no definitive invasive adenocarcinoma was seen. PancreaSeq GC has a cystic precursor score of 6 and a risk score of 5 (Supplementary Table 1, Supplemental Digital Content 1, http://links.lww.com/SLA/A622, Case 28).

FIGURE 2

An oncoplot of 108 pancreatic cyst fluid specimens that were used for PancreaSeq GC training and includes cyst type, associated clinical features, and DNA/RNA-based sequencing results. At the bottom is a summary of genomic alterations involving MAPK gene, GNAS, and/or RNF43 alterations; cystic precursor score; summary genomic alterations involving TP53, SMAD4, mTOR genes, and CTNNB1; and risk score.

FIGURE 3

An oncoplot of 77 pancreatic cyst fluid specimens that were used for PancreaSeq GC validation and includes cyst type, associated clinical features, and DNA/RNA-based sequencing results. At the bottom are the corresponding cystic precursor and risk scores.