PancreaSeq® Genomic Classifier - Pancreatic Cyst Fluid NGS Analysis
PancreaSeq® Genomic Classifier (GC) is a diagnostic test for pancreatic cysts detected by imaging, including benign cysts (e.g., serous cystadenomas), mucinous cysts (IPMN/MCN), cystic pancreatic neuroendocrine tumors (PanNETs), and early detection of pancreatic ductal adenocarcinomas.
PancreaSeq GC test is DNA and mRNA next-generation sequencing analysis of 74 genes. The method allows to detect single nucleotide variants (SNVs), insertions and deletions (indels) in 20 genes related to pancreatic cysts and pancreatic cancer, including KRAS and GNAS, loss of heterozygosity (LOH) analysis and copy number alterations (CNAs) at 13 chromosomal regions, including the RNF43, SMAD4, TP53, PTEN, VHL, and NF2 tumor suppressor genes. In addition, it detects >150 types of gene fusions involving the ALK, BRAF, FGFR2, NTRK1, NTRK3, RET, ROS1, RAF1, PRKCA, PRKCB genes and expression of specific genetic markers, including KRT7, KRT20, CHRGR, and PGK1. Finally, it detects expression of the CEA (CECAM5) gene by qRT-PCR. The test is using a small amount of nucleic acids (DNA and RNA) isolated from pancreatic cyst fluid collected into preservative solution during ultrasound-fine needle aspiration (EUS-FNA) procedure. The findings are subjected to algorithmic analysis to categorize as Negative or Positive for specific alterations associated with different cyst types and risk of progression.
Both pancreatic cysts and pancreatic solid lesions represent a broad and diverse group of benign and malignant entities. Among pancreatic cysts, distinguishing one pancreatic cyst from another can be challenging on the basis of standard clinical findings, imaging parameters and ancillary fluid studies, such as cytology and CEA analysis. DNA sequencing studies of pancreatic cysts have identified a limited number of genetic alterations that can be used diagnostically and prognostically to classify pancreatic cysts.(1-4, 6-8) Intraductal papillary mucinous neoplasms (IPMNs) and mucinous cystic neoplasms (MCNs) represent mucinous pancreatic cystic neoplasms. Over 95% of IPMNs are characterized by mutations in the genes: KRAS (codons 12, 13 and/or 61), GNAS (codons 201 and 227), RNF43, BRAF, and CTNNB1. (1-4, 30, 31) KRAS, RNF43 and CTNNB1 mutations can also be found in MCNs with a prevalence that ranges from 14% to 50%.(1-4, 6-7, 9) In contrast to IPMNs, MCNs do not harbor GNAS and BRAF mutations, and, thus, genetic alterations in GNAS and BRAF are highly specific for IPMNs.(4, 7, 30) Other neoplastic cysts include serous cystadenomas and solid pseudopapillary neoplasms. Serous cystadenomas (SCAs) have an extremely low malignant potential and approximately 89% to 100% harbor mutations and/or deletions in VHL, but lack mutations in KRAS, GNAS and BRAF.(1-3, 7, 30) Finally, solid-pseudopapillary neoplasms (SPNs) are characterized by the presence of CTNNB1 mutations (within exon 3), and an absence of alterations in KRAS, GNAS, RNF43, BRAF and VHL.(3, 7)
IPMNs and MCNs are precursor neoplasms to pancreatic ductal adenocarcinoma; however, only a subset harbor or progress to malignancy. Studies have shown that IPMNs and MCNs with genetic alterations in TP53, SMAD4 and the phosphatidyl-3 kinase (PI3K) pathway, which include PIK3CA, PTEN, and AKT1, are associated with high-grade dysplasia and early invasive pancreatic ductal adenocarcinoma (advanced neoplasia). Kanda et al detected TP53 mutations in 56% of IPMNs with advanced neoplasia.(10) Similarly, 40% to 60% of IPMNs with advanced neoplasia harbor alterations in PIK3CA, PTEN, AKT1 and SMAD4.(11-13) Using EUS-FNA obtained pancreatic cyst fluid, Singhi et al found 88% of IPMNs with advanced neoplasia have mutations in KRAS and/or GNAS with concurrent alterations in TP53, PIK3CA, PTEN or AKT1.(1-2)
Cystic pancreatic neuroendocrine tumors (PanNETs) are typically diagnosed by standard cytology, but the diagnosis may be facilitated by the presence of MEN1 and/or TSC2 mutations in a subset of these pancreatic cysts. Genetic alterations are absent in benign non-neoplastic cysts, such as pseudocysts, lymphoepithelial cysts, retention cysts, squamoid cysts or acinar cell cystadenomas.(1-3) However, adequate sampling and preservation of the specimen should always be considered when evaluating molecular testing results. And therefore, correlation of molecular testing with cytology, imaging and other clinical data is recommended. It is important to underscore that several of the aforementioned genetic alterations have been discussed by the International Consensus Fukuoka Guidelines for the management of IPMNs and MCNs, and the European Evidence-Based Guidelines on pancreatic cystic neoplasms.(15-16) Both guideline organizations highlight the utility of these DNA markers in the diagnosis of pancreatic cysts.
In addition, solid pancreatic lesions have a wide range of pathology, from chronic pancreatitis to pancreatic ductal adenocarcinoma (PDAC). Endoscopic ultrasound (EUS) with fine-needle aspiration (FNA) is an important diagnostic tool in the work up of a solid pancreatic lesion with sensitivities as high as 80% to 95% and specificities as high as 75% to 100%.(17-19) However, in a subset of cases, the preoperative diagnosis remains inconclusive due to limited cellularity, leading to a nondiagnostic, atypical or suspicious cytopathologic diagnoses.(20,21)
Next-generation sequencing (NGS) has been instrumental in our understanding of the genome of various solid lesions of the pancreas and can be used as an adjunct to the evaluation of solid pancreatic lesions.(22,23) For example, PDAC is characterized by frequent genomic alterations in KRAS, TP53 and/or SMAD4. Kameta et al demonstrated that NGS for KRAS, TP53 and SMAD4 alterations on EUS-FNA specimens is associated with a 96%, 44% and 11% sensitivity, respectively, and 100% specificity for PDAC.(24) Similarly, Young and colleagues found EUS-FNA specimens harboring mutations in KRAS, TP53 and/or SMAD4 were present in 95% of cases that correlated with PDAC.(25) Within a large cohort of EUS-FNA specimens, Gleeson et al. found KRAS, TP53 and SMAD4 alterations were present in 93%, 72% and 31% of PDACs.(26)
In contrast to PDAC, pancreatic neuroendocrine tumors (PanNETs) do not have KRAS mutations, but harbor frequent alterations in MEN1, VHL, and/or TSC2.(23, 14) Further, recurrent genomic alterations in several chromatin remodeling genes leads to numerous chromosomal copy number alterations, which is associated with decreased disease-free survival and decreased disease-specific survival.(14, 27) This is especially critical when evaluating small neuroendocrine tumors (27). Moreover, these prognostic findings can be extended to other neuroendocrine tumors of the gastrointestinal tract, such as those found in the colon, small intestine and stomach, and hence studies strongly support the utility of molecular profiling of all gastrointestinal tract well-differentiated neuroendocrine tumors.(28, 29)
A pancreatic cyst fluid carcinoembryonic antigen (CEA) is a useful marker in identifying mucinous cysts. (34) The CEACAM5 gene encodes a cell surface glycoprotein that plays a role in cell adhesion, intracellular signaling and tumor progression and is the founding member of the carcinoembryonic antigen (CEA) family of proteins. Measuring mRNA expression of the CEACAM5 gene in pancreatic cyst fluid samples can be used to detect CEA upregulation. (32, 33)
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- Singhi AD, et al. Mod Pathol. 2020;33:1739-1801.
- Fischer CG, et al. Gastroenterology. 2019;157:1123-1137.e22.
- Vuijk FA, et al. Sci Rep. 2020;10:16211; 33. de Albuquerque A, et al. Clin Lab. 2012;58:373-84; 34. Khan I, et al. Dig Dis Sci. 2021.
Examples of PancreaSeq GC Reports
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