An appraisal of pancreatic cyst fluid molecular markers
Rohan M. Modi, Ravi B. Pavurala, Somashekar G. Krishna
Abstract
Pancreatic malignancy is the third leading cause of cancer related death in the United States with limited viable screening options. By the end of this decade, cancers are poised to become the leading cause of death with pancreatic cancer projected to be the second leading cause of cancer related mortality. Pancreatic cystic lesions (PCLs) are found in approximately 5%–14% of patients due to the increased utilization of cross-sectional imaging, with approximately 8%–10% of pancreatic cancers originating as PCLs. Current screening guidelines have shown discrepancies between morphologic characteristics of PCLs and identifying advanced pancreatic disease. Molecular analysis has emerged as a novel technology to aid in adequate diagnosis and management decisions of PCLs. Mucinous cysts including intraductal papillary mucinous neoplasms (IPMNs) or mucinous cystic neoplasms have similar oncogenic mutations including KRAS, TP53, SMAD4, PIK3CA, PTEN, or CKDN2A, while GNAS and RNF43 mutations are specific only to IPMNs. Serous cystadenomas have been associated with a loss of tumor suppressor gene VHL, while solid-psuedopapillary neoplasms have an oncogenic mutation CTNNB1. A specific molecular marker to diagnose existing high-grade dysplasia or impending malignant transformation is yet to be identified. Moving forward it is important to advance technology in isolating and identifying high-risk molecular markers from cyst fluid while considering their increased utilization in the evaluation of PCLs.
Introduction
Pancreatic cancer has risen to be the third leading cause of mortality secondary to malignancies in the United States with an estimated mortality of 41,780 people for the year 2016.1 Moreover, the 5-year survival for pancreatic cancer is alarmingly low at 6% and has been unchanged over the past 40 years.2 While we have limited viable screening options for pancreatic cancer, increased utilization of cross-sectional abdominal imaging has resulted in an increase number of pancreatic cystic lesions (PCLs) being discovered. In fact, PCLs are found in approximately 2%–14% of patients who undergo either computed tomography (CT) or magnetic resonance imaging (MRI).3–5 The influx of patients with undifferentiated PCLs has outlined the significance of determining which precursor lesions will progress to pancreatic adenocarcinoma or metastatic disease.6
Guidelines for Screening Pancreatic Cystic Lesions
The current approach for evaluating a PCL is multidisciplinary including radiographic findings, endoscopic ultrasound (EUS), fine needle aspiration (FNA), tumor markers (carcinoembryonic antigen [CEA]), and cytology. The screening approach over the past decade has been outlined by International Consensus Guidelines (2007 Sendai, 2012 Fukuoka), and the American Gastroenterology Association (AGA) (Table 1).7–9 The Sendai guidelines were introduced in 2006 and recommended surgical removal for any suspected MCN, main duct intraductal papillary mucinous neoplasm (MD-IPMN), and mixed duct IPMN.7 Additional criteria for surgery included the following: clinical symptoms, dilated pancreatic duct (≥ 6 mm), intracystic mural nodule, or positive cytology. While these guidelines had a sensitivity of 100%, multiple studies indicated low specificity ranging from 23%–31%.10,11 Subsequently, revised Fukuoka guidelines were presented in 2012 with stricter surgical criteria: pancreatic duct ≥ 10 mm, an enhancing solid component, or obstructive jaundice with a pancreatic head cyst.8 While this resulted in improved specificity, there was a loss in sensitivity to detect advanced neoplasia and no statistical differences when compared to the Sendai guidelines.12
Both Sendai and Fukuoka guidelines are limited as they only focus on IPMN and MCN cysts. The introduction of AGA guidelines in 2015 focused on having a uniform approach for all asymptomatic neoplastic cysts. Compared to Fukuoka, AGA guidelines presented a more rigorous standard for both EUS-FNA and surgical removal that included two high-risk features: size > 3 cm, presence of mural nodule, and dilated main pancreatic duct in the setting of positive cytology.9 The stringent criteria for endoscopic evaluation of PCLs has resulted in concern that following AGA guidelines would result in inadequate identification of progressive neoplastic disease. One retrospective study found that using AGA guidelines on a cohort of 225 patients had lower sensitivity and specificity of 62% and 79% respectively for detecting advanced neoplasia.13 Moreover, the guidelines would have missed 45% of IPMNs with adenocarcinoma or high-grade dysplasia.
Collectively these guidelines indicate that morphologic characteristics are inadequate predictors of advanced pancreatic disease. The introduction of molecular analysis has emerged as a novel technology to assist in the evaluation of PCLs prior to surgical intervention.14,15 The progression from a premalignant cyst to advanced neoplasm can occur through a variety of mechanisms including oncogene mutations, loss of tumor suppression genes, or hypermethylation. While cytologic testing requires optimal levels of pancreatic cyst fluid, the benefit of molecular testing is that minimal aspirate is required from the PCL to assess for genetic alterations. Our aim in this review is to summarize the current literature on distinct gene mutations within subtypes of PCLs for possible integration in diagnostic evaluation of cystic lesions.
Classification of Pancreatic Cystic Lesions
The four most common PCLs include intraductal papillary mucinous neoplasm (IPMN), mucinous cystic neoplasm (MCN), serous cystadenoma (SCA), and solid pseudopapillary neoplasm (SPN). IPMNs are most commonly seen in older individuals with the majority located in the pancreatic head or uncinate with expected communication with the main pancreatic duct, while MCNs are typically seen in middle-aged women (40s–50s) primarily in the body or tail of the pancreas. Both IPMNs and MCNs are mucinous cystic lesions and are likely to have elevated CEA in cystic fluid. On the other hand, SPNs are seen in younger women (20s–30s) who present with symptoms with a slight predisposition for the pancreatic body or tail, while SCAs are usually present in older women and are similarly found in the body or tail of the pancras.16
Because the aforementioned characteristics are generalizations, the challenge is an accurate identification of PCL type to adequately guide management decisions. For example, SCAs are benign, non-mucinous lesions that do not require surgical removal or imaging surveillance unless clinically warranted.17,18 On the other hand, SPNs are classically seen in young women and given their aggressive metastatic nature are surgically resected.16,19 Similarly, MCNs can progress to invasive disease and are usually resected rather than continued imaging or endoscopic surveillance.8 Finally, IPMNs are followed due to slow progression from low or intermediate to high or invasive adenocarcinoma.20,21 While low or intermediate grade dysplasia can be monitored through surveillance, more advanced lesions needs to be surgically resected.8 Other rare types of PCLs including cystic pancreatic neuroendocrine tumors (cystic-NETs), squamous lined cysts (lymphoepithelial cysts, epidermoid cysts), and cystic degeneration of metastatic cancers pose additional challenges and diagnosis relies significantly on EUS-FNA cytology.16 The aforementioned management decisions demonstrate the importance of differentiating PCL type with eventual goal of preventing development of pancreatic malignancy.
Genetic Variation between Pancreatic Cystic Lesions
Intraductal papillary mucinous neoplasm
IPMNs are stratified based on the involvement of the main pancreatic duct and include MD-IPMN, branch duct IPMN (BD-IPMN), or mixed IPMN. This classification is important as a recent analysis of the IPMN literature reported malignancy incidence between 57%–92% when the main pancreatic duct is involved (MD-IPMN, mixed IPMN) and 6%–46% in BD-IPMN.22 Furthermore, IPMNs can be characterized on the basis of morphology including gastric, intestinal, pancreatobiliary and oncocytic subtypes.22 Previous molecular studies have focused on DNA quantity and quality, KRAS/GNAS, and LOH with varying results. A 2009 study compared CEA levels and DNA quantity and found DNA quantity had a 76% sensitivity and 100% specificity for the diagnosis of a mucinous cyst. However, when CEA and molecular analysis were combined, a sensitivity of 100% was achieved.23
In terms of oncogenic mutations, both KRAS and GNAS have been studied extensively in IPMNs. KRAS codes for a guanosine-nucleotide binding protein and mutations in KRAS codon 12 are present in approximately 90% of all pancreatic ductal carcinomas.24 Various studies have shown that KRAS has excellent specificity (ranging from 96%–100%), but has limited sensitivity (ranging from 45%–65%) in detecting mucinous neoplasms.25–27 When combining CEA and KRAS, one study noted an increase in identification in pre-malignant and malignant cysts.26 On the other hand, GNAS causes downstream production of cAMP that ultimately results in uncontrolled growth.28GNAS mutations are more prevalent in the intestinal IPMN subtype (71%) when compared to gastric (50%) or pancreatobiliary (31%) subtypes. Additionally, the presence of GNAS or KRAS seem to occur early in IPMN carcinogenesis as these mutations had no variability based on degree of dysplasia.28 A 2013 study found GNAS mutation was seen in 44% of IPMNs and that both KRAS and GNAS had a combined sensitivity of 60%.29 The advent of massive parallel sequencing, also known as next-generation sequencing (NGS) or deep sequencing where accurate detection of mutations in gene panels with limited DNA can be achieved, has demonstrated that both GNAS and KRAS mutations are found in 92% of all IPMNs.30
Several studies have combined DNA quantity, KRAS mutations, and LOH and have shown variable sensitivities: 50%31 vs 83%.32 An additional study found that both KRAS and LOH was present in 50% of carcinoma or high grade dysplasia compared to 8% of premalignant IPMNs, indicating the progression of neoplasia may be correlated by increased number of genetic disturbances.33 Additional molecular markers present in IPMNs include p16 (lost earlier compared to p53), SMAD4, p53, RNF43, and TP53 (Table 2).33–36
Mucinous cystic neoplasm
MCNs are typically seen in middle-aged women and located in the body or tail and are not associated with the main pancreatic duct.16 These cysts are lined by mucin producing tall columnar epithelial cells with mild to severe dysplasia. Based on the International consensus guidelines, MCNs were differentiated from IPMNs based on presence of ovarian stroma.37 Unlike IPMNs, the incidence of KRAS mutations increases with the severity of dysplasia in MCNs. This was outlined by a study showing KRAS detection in 20% benign, 33% borderline, and 89% of malignant MCN lesions.38 However, a recent study indicated KRAS mutations have a sensitivity of 14% detecting MCN lesions, which is significantly lower than its IPMN counterpart.27 This finding was replicated again in 2014 where only 1/16 patients (6%) had a positive KRAS mutation in an MCN population. Multiple studies have ruled out GNAS as a potential marker for MCNs as this mutation has only been found in IPMN lesions.39 The genetic alterations in MCNs include KRAS, TP53, and SMAD4. Additional associations with PIK3CA, PTEN, and CKDN2A have also been published.40–42 However, the lack of sensitivity of KRAS portends the continued need for research to ensure accurate identification of MCNs.15
Serous cystadenoma
SCAs are benign cystic neoplasms that generally present in older women with a slight predisposition for the body or tail of the pancreas. The neoplasm is a multilobular cyst with a thin capsule that can have radiographic findings such as sunburst pattern of calcification. SCAs consist of single-layer epithelium with glycogen-rich clear cells within cysts.43 Given their indolent slow growth and only 28 cases of serous cystadenocarcinomas in the past 3 decades, the current management of asymptomatic patients is continued surveillance without surgical intervention.18 Molecularly, mutations seen in tumor suppressor gene VHL and LOH of chromosome 3 were both associated with SCAs.41,42 The VHL gene is important as it encodes for an E3 ubiquitin ligase that inhibits activation of HIF1-a, an oncogene. In the absence of the E3 ubiquitin ligase, HIF1-a can promote cell proliferation and angiogenesis.2,41 The combination of VHL + LOH of chromosome 3 resulted in a sensitivity of 100% and specificity of 91%; but combining molecular and clinical markers increased the specificity to 98%.42 Finally, it is important to note that in stark contrast to IPMNs or MCNs, these cysts do not have mutations of KRAS, GNAS, RNF43, TP53, SMAD4, PIK3CA, PTEN, or CKDN2A.
Solid pseudopapillary neoplasm
SPNs are typically well-demarcated solitary lesions that are found in younger women (mean age of 25 years).19 SPNs have variable features on CT/EUS and can be cystic, solid, or have both cystic and solid components. These lesions do not communicate with the main pancreatic duct and consist of branching papillae with myxoid stroma on cytology.16 Molecular analysis of SPN has fewer genetic alterations compared to other cysts, but always have a missense mutation CTNNB1, present at codon 32, 33, 34, or 37.41 Other mutations such as TP53 and PIK3CA are rarely found, while KRAS, GNAS, RNF43, CDKN2A, SMAD4, and VHL mutations are never found. Therefore, the presence of CTNNB1 and absence of KRAS, GNAS, and RNF43 mutations has a 100% sensitivity and specificity in detecting SPNs.42
Other pancreatic cystic lesions
Other rare types of PCLs including cystic-NET, squamous lined cysts including lymphoepithelial cysts and epidermoid cysts of accessory spleen, and secondary degeneration of solid primary/ metastatic lesions to the pancreas have not been characterized by molecular studies. Cystic-NETs represent approximately 8% of resected PCLs and are more common than SPTs (4% of resected PCLs).44 A majority of cystic-NETs are indistinguishable from other PCLs on preoperative imaging and cyst-fluid CEA levels are generally very low; however, the yield of EUS-FNA cytology is high (73%) compared to other PCLs (20%).44,45
Integration of Molecular Testing in the Management of Pancreatic Cysts
Cyst fluid molecular markers can play an integral role in not only differentiating the types of PCLs, but also in risk-stratification of mucinous lesions. This facet of research will widen further with the management of PCLs becoming more conservative. Future guidelines will need to decide how to integrate molecular testing into a working algorithm for all PCLs. Recently, the University of Pittsburgh Medical Center based on PanSeq NGS Panel has proposed their own pathway in which there is a lower threshold to perform an EUS-FNA and they have three criteria to consider surgical removal: (1) positive or suspicious cytopathology for malignancy or (2) mucinous cysts > 3 cm with either main pancreatic duct dilation or definitive mural nodule or (3) molecular testing including KRAS and/or GNAS with either TP53 or PIK3CA/PTEN.2 Albeit an encouraging step towards introducing molecular analysis into common practice, studies will need to be performed to validate such a drastic deviation from current guidelines. The gene-panel for evaluating PCLs is also evolving with a very recent study utilizing a 39-gene panel (compared to 7 genes of PanSeq NGS Panel) to demonstrate a discordance rate of 22% between clinical cyst characterization and subsequent NGS examination.46
Future studies should focus on molecular markers to improve sensitivity of MCNs to ensure adequate management of these PCLs. It is of concern that KRAS mutations have limited accuracy and that there are no other reliable oncogenic mutations or tumor suppressor genes that have been commonly associated with MCNs. Reliable molecular markers to differentiate low versus high grade IPMN is another challenge with current analytic methods. EUS-guided needle based confocal laser endomicroscopy (nCLE) can characterize common types of PCLs as demonstrated in several recent studies and the image patterns have been validated.47–52 A combined evaluation of PCLs with EUS-nCLE and molecular analysis of cyst fluid could possibly provide a more accurate diagnostic pathway for effective patient management.
Conclusion
PCLs are increasingly detected on cross-sectional imaging studies. The international consensus guidelines indicate that morphologic characteristics are unable to definitely determine advanced neoplasia in cystic lesions. Molecular analysis has emerged as a novel technology to aid in adequate diagnosis and risk stratification of PCLs. Although the diagnostic parameters and techniques for detection of molecular markers are rapidly evolving and increasingly utilized in practice, there is a need for more definitive evidence of their clinical value for wide-spread utilization and definitive incorporation in management guidelines of PCLs.
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