Context.—

Integration of the analysis of genetic markers with endoscopic ultrasound–guided fine-needle aspiration and cytologic evaluation has increased the accuracy of the preoperative diagnosis of pancreatic lesions. The application of high-throughput gene panel analysis using next-generation sequencing platforms is now offering a great opportunity for further improvements.

Objective.—

To review the application of next-generation sequencing to the preoperative diagnosis of pancreatic lesions.

Data Sources.—

For data acquisition, a PubMed search using the terms next-generation sequencing, pancreas, pancreatic lesions, pancreatic tumors, and EUS-FNA was performed covering the years 2000–2017.

Conclusions.—

KRAS remains the gene most widely studied for preoperative single-gene tests. Next-generation sequencing reliably allows analysis of multiple gene markers starting from limited amounts of DNA. The study of multigene panels has become a very attractive option for the management and preoperative risk stratification of patients with pancreatic cancer.

P ancreatic ductal adenocarcinoma (PDAC) is one of the most aggressive types of cancer in humans and is usually diagnosed at an advanced stage.1  The management of patients with a pancreatic mass (tumor or cysts) is still a challenge. Endoscopic ultrasound (EUS)–guided fine-needle aspiration (FNA) has greatly improved preoperative diagnosis,2  with sample adequacy (ie, the acquisition of diagnostic material) currently ranging from 65% to 96%.3,4  Although EUS-FNA plus cytologic evaluation shows high clinical sensitivity (calculated according to recommendations previously described5 : true-positive/[true-positive + false-negative]) and specificity (true-negative/[true-negative + false-positive]), in a subset of cases the preoperative diagnosis remains inconclusive because of inadequate/insufficient material or limited cellularity, leading to atypical/suspicious cytopathologic diagnoses.6 

In most cases, PDAC is initiated by oncogenic mutant KRAS, which has been shown to drive pancreatic neoplasia.7,8  In the past 2 decades several studies have shown how the analysis of KRAS mutations of pancreatic lesions improves diagnostic accuracy and is particularly useful in those cases where EUS-FNA cytology is inconclusive.914 

Next-generation sequencing (NGS) has been instrumental for the understanding of the pancreatic cancer genome. Whole-exome sequencing of 20 661 genes has shown that any given PDAC contains an average of 63 genomic alterations, most of which are point mutations.15  High-throughput molecular analysis has demonstrated that in addition to KRAS mutation, inactivations of TP53, SMAD4, and CDKN2A/p16 are the pivotal molecular alterations that define the development and progression of PDAC.16,17  This finding, and the variety of molecular alterations found in different types of pancreatic tumors (summarized in Table 1), such as CTNNB1 mutations in solid pseudopapillary neoplasm (SPN) or GNAS mutations in intraductal papillary mucinous neoplasm (IPMN), underscore the need for the analysis of multiple biomarkers.1821 

Table 1

Main Genetic Alterations in Pancreatic Tumors

Main Genetic Alterations in Pancreatic Tumors
Main Genetic Alterations in Pancreatic Tumors

The introduction of NGS to molecular diagnostics has allowed performance of molecular analysis of multiple genes in limited samples, opening new avenues to the study of preoperative specimens. Preoperative cytologic smears (ie, material obtained during FNA or biliary brushings smeared on a slide) and fine-needle biopsies (ie, small samples obtained using a 22-gauge coring needle that provides cylindrical specimens for the examination of tumor architecture) are characterized by a small quantity of diagnostic material, and are often composed of heterogeneous cell populations. For this reason, it is crucial to use molecular tests that offer high analytical sensitivity to detect small proportions of mutated cells. Next-generation sequencing combines high analytical sensitivity with multiple gene analysis and thus represents a very attractive option.

The scope of this review is to provide an updated survey of the studies that have used NGS for the preoperative molecular workup of pancreatic lesions. To identify the pertinent references, a PubMed search using the terms next-generation sequencing, pancreas, pancreatic lesions, pancreatic tumors, and EUS-FNA was performed covering the years 2000–2017. Our review addresses studies that analyzed (1) solid pancreatic lesions, (2) pancreatic cyst fluid, or (3) other fluid specimens from patients with pancreatic tumors.

The studies that addressed NGS analysis of EUS-FNA samples of solid lesions are summarized in Table 2. In 2014, De Biase et al13  demonstrated that NGS is more sensitive and specific than conventional mutation analysis for the preoperative identification of pancreatic lesions with malignant potential. The authors observed that using NGS to analyze KRAS mutations in pancreatic FNA specimens allows clinical sensitivity to increase up to ∼74%, a value far superior to that obtained by allele-specific real-time polymerase chain reaction (PCR) (52.8%) or by Sanger sequencing (42.1%), while maintaining clinical specificity at 100%. The analytical sensitivity was higher if the analysis was performed starting from cells scraped from the smear preparations used for routine cytologic diagnosis, as opposed to FNA material directly submitted by the endoscopist for molecular diagnosis.13  Targeted KRAS parallel sequencing had higher clinical sensitivity when compared with commercially available KRAS mutation–specific diagnostic kits.22 

Table 2

Next-Generation Sequencing Analysis of Solid Pancreatic Tumors Starting From Fine-Needle Aspiration (FNA) Material

Next-Generation Sequencing Analysis of Solid Pancreatic Tumors Starting From Fine-Needle Aspiration (FNA) Material
Next-Generation Sequencing Analysis of Solid Pancreatic Tumors Starting From Fine-Needle Aspiration (FNA) Material

Kameta et al23  analyzed a panel of 50 most commonly mutated oncogenes (Ampliseq Cancer Hotspot Panel v2.0, designed to amplify 207 amplicons covering ∼2790 mutations; Thermo Fisher Scientific, Waltham, Massachusetts) in a cohort of 38 patients with pancreatic disease, 27 of whom were diagnosed with PDAC. The sequencing was performed on DNA extracted from EUS-FNA material and showed that KRAS was the most frequently mutated gene (96%; 26 of 27 PDACs). TP53 mutations were detected in 44% of the samples (12 of 27) and both SMAD4 and CDKN2A in 11% (3 of 27). Intriguingly, 2 mutations detected by NGS were not detected by the real-time PCR assay used by the authors for comparison (presumably because the mutation-specific real-time PCR assay was not designed to detect them). The authors also analyzed samples from metastatic tumors of unknown origin to investigate if the panel might be of help to identify the primary lesion, but the results were limited by the lack of specificity of KRAS mutations, which were commonly present not only in PDAC but also in adenocarcinomas of lung, colon, and stomach. They concluded that their assay could be used for the preoperative diagnosis of pancreatic EUS-FNA, even if a 50-gene panel might not be sufficient to fully profile the molecular landscape of PDAC.

Young and colleagues24  tested a large multigene panel (287 cancer-related genes, covering a total of 4561 exons and 47 introns of 19 genes frequently rearranged in cancer) on 23 pancreatic FNAs. The cohort consisted of 17 PDACs, 3 mucinous adenocarcinomas, 2 adenocarcinomas not otherwise specified, and 1 neuroendocrine tumor. The authors observed a mean of 3.8 mutations per tumor (range 1–9). The most common alterations were detected in KRAS (78%; 18 of 23 cases), TP53 (74%; 17 of 23), CDKN2A/B (35%; 8 of 23) and SMAD4 (17%, 4 of 23). Mutations of PTEN were found in 13% of samples. The comparison of molecular analysis performed on FNA and on matched surgical specimens showed a perfect concordance of the mutational profile between the two.

In a study of 29 EUS-FNA samples, Gleeson and colleagues25  used a 160-gene panel to preoperatively determine the mutational landscape of 21 PDACs, 4 ampullary carcinomas, 1 Lynch syndrome–associated PDAC (not previously treated with chemotherapy) and 3 IPMNs, and to verify its concordance with that obtained from surgical resection specimens. They demonstrated that a moderate to large targeted NGS analysis of preoperative EUS-FNA samples provides an excellent surrogate for the analysis of surgically resected specimens to detect mutations associated with pancreatic cancer. The main genes altered were KRAS (93%; 27 of 29), TP53 (72%; 21 of 29), SMAD4 (31%; 9 of 29), and GNAS (10%; 3 of 29). A TP53 mutation was observed frequently in association with a KRAS alteration (95%; 20 of 21 TP53-mutated cases). Pathogenic SMAD4 alterations were detected in about 30% of patients with PDAC (7 of 21), but in none of the patients with ampullary carcinoma. They reported an excellent concordance between mutations found in EUS-FNA specimens and those detected in the paired surgical material: in 15 of 18 cases the concordance was 100%. In 3 samples, they observed some mutations in EUS-FNA specimens (in GRIN2A, GATA3, GNAS, and KDM6A genes) but not in the surgical specimens: all discordant mutations had a percentage of mutated alleles lower than 14% and were therefore consistent with subclonal molecular events. Genetic heterogeneity (including heterogeneity of KRAS mutations, an early initiating event) has been reported in pancreatic cancers.26,27  Considering that preoperative samples contain only a small part of the lesion, this represents a limitation of all preoperative diagnostic procedures (including conventional cytologic examination) in pancreatic cancers as well as in cancers from other organs. The proportion of mutated alleles identified by NGS represents an important clue to define a mutation as subclonal. It can be normalized to estimate the proportion of neoplastic cells carrying the mutation28  and should be taken into account for molecular diagnosis. In the work by Gleeson and colleagues,25  as well as in our experience, molecular heterogeneity represented a relevant issue in only a minority of pancreatic tumors.

Sibinga Mulder and colleagues29  also reported a good correlation between mutations detected in preoperative FNA material and those found in the matched surgical resection specimen.29  Using a panel of 50 genes frequently mutated in cancer for the profiling of FNA material from the PDAC of a 54-year-old man, they detected mutation in KRAS, TP53, SMAD4, and CDKN2A starting from both preoperative cytologic and surgical resection specimens.

Next-generation sequencing analysis has also been successfully performed from pancreatobiliary brush cytology samples, as demonstrated by Dudley and colleagues30  in a cohort of 81 patients who underwent endoscopic retrograde cholangiopancreatography. Also in this cohort of samples, KRAS mutations were the most frequent alteration (26%; 21 of 81 cases analyzed by NGS), and TP53 was the second most commonly mutated gene (17%; 14 of 81 cases). The authors30  observed that NGS is as sensitive as the analysis of aneuploidy by fluorescence in situ hybridization to preoperatively identify pancreatobiliary duct malignancies. Intriguingly, in this paper KRAS mutations were detected also in 2.3% (1 of 43) of nonneoplastic control samples.30  It should be noted that KRAS mutations have been reported in some cases of chronic pancreatitis, where the presence of KRAS mutation has been associated with evolution of the pancreatitis to PDAC.31,32 

KRAS (74%; 14 of 19 cases), TP53 (47%; 9 of 19 cases), and SMAD4 (32%; 6 of 19 cases) were also the most frequently mutated genes in the study by Valero and colleagues33  performed on a cohort of 19 FNAs from patients with unresectable nonmetastatic pancreatic tumors, using a panel of 409 cancer-related genes. The authors also found mutations in ARID1 (16%; 3 of 19 cases), GRM8 (10%; 2 of 19 cases), and TRIM33 (10%; 2 of 19 cases). They concluded that somatic variants identified in preoperative FNA samples using NGS may be used to guide the clinical management of patients with pancreatic cancer.

Differently from what is observed in PDAC, SPNs do not have KRAS mutations, but harbor mutations in CTNNB1, the gene of the wnt pathway encoding for β-catenin. Kubota et al34  investigated CTNNB1 using NGS in a cohort of SPNs, PDACs, and pancreatic neuroendocrine tumors (P-NETs) starting from EUS-FNA material. Mutations of CTNNB1 were detected in all SPNs, 9% (1 of 11) of pancreatic neuroendocrine tumors, and no PDAC specimens. Even if the percentage of mutated alleles detected by NGS was 20% or more (a percentage compatible with the analytical sensitivity of Sanger sequencing), they were able to identify CTNNB1 mutation in only 1 of the samples using Sanger sequencing.

The studies that have addressed NGS analysis of EUS-FNA samples of cystic lesions are summarized in Table 3. Pancreatic cysts are a heterogeneous group of lesions that include injury and inflammation–related conditions (∼30% of cases) as well as neoplasms (∼60% of cases). The large majority of neoplastic cysts are of ductal lineage, more frequently mucinous, IPMN, and mucinous cystic neoplasm (MCN), but also of serous lineage (serous cystadenoma and rarely cystadenocarcinoma).35 

Table 3

Next-Generation Sequencing Analysis of Cystic Pancreatic Lesions Starting From Fine-Needle Aspiration (FNA) Material

Next-Generation Sequencing Analysis of Cystic Pancreatic Lesions Starting From Fine-Needle Aspiration (FNA) Material
Next-Generation Sequencing Analysis of Cystic Pancreatic Lesions Starting From Fine-Needle Aspiration (FNA) Material

Al-Haddad and colleagues36  have shown that the analysis of KRAS and loss of heterozygosity helps in the differential diagnosis of cystic mucinous pancreatic lesions (IPMN and MCN) when preoperative cytology is nondiagnostic or carcinoembryonic antigen (CEA) cyst fluid levels are indeterminate. The biochemical determination of CEA is one of the most accurate tumor markers to diagnose mucinous pancreatic cysts and to distinguish them from nonmucinous cysts: in fact, high levels of CEA (>200 ng/mL) strongly suggest mucinous neoplasia although reported cutoff CEA values vary considerably.37,38 

Several studies have now demonstrated that high-throughput analysis of multiple genetic markers with NGS platforms adds useful information to that obtained after the evaluation of CEA and cytologic specimens.

Amato et al39  analyzed 51 cancer-associated genes using NGS in 48 IPMNs. The marker more commonly altered was GNAS (79%; 38 of 48 cases). KRAS was mutated in 50% of IPMNs (24 of 48), and in 37.5% (18 of 48) the mutation coexisted with GNAS alterations. Less frequently, mutations were found in TP53 (10%; 5 of 48 cases), BRAF (6%; 3 of 48 cases), and CTNNB1 and IDH1 (4%; 2 of 48 cases for each gene). KRAS and GNAS mutations coexisted in 37.5% of IPMNs (18 of 48). The amount of DNA obtained from cyst fluid was adequate for NGS (ie, at least 20 ng of DNA) in 10 of 48 IPMNs. In these 10 samples, sequencing allowed detection of 10 of the 13 mutations found in the matched surgical specimens (6 of 7 GNAS mutations, 3 of 3 KRAS mutations, and 1 of 2 TP53 mutations).39  The combination of GNAS and KRAS preoperative testing was also demonstrated to be highly specific and sensitive for IPMNs by Singhi et al.40 

Jones et al41  analyzed 92 cyst fluid samples using a panel of 39 cancer-related genes. KRAS was the gene most frequently mutated (47%; 43 of 92 cases), followed by GNAS (24%; 22 of 92 cases) and CDKN2A (7%; 6 of 92 cases). Overall, 43% (40 of 92) of the samples did not show any mutation in at least 1 of the 39 genes in the panel. In 71% (65 of 92) of the samples a KRAS or GNAS mutation was consistent with a diagnosis of IPMN by imaging, in spite of low CEA levels. In one case, an elevated level of CEA was discordant with the impression based on imaging, but the finding of one KRAS mutation supported the preoperative diagnosis of MCN.41  The data of the study supported the association between high-risk cysts and accumulation of genetic alterations. KRAS analysis provided useful information for the malignancy risk in those cases that would be diagnosed as benign by imaging and in those with low CEA levels in the cyst fluid. The use of a panel of genes helped in detecting those mutations typically associated with mucinous lesions (KRAS, GNAS, CDKN2A) and those additional changes in genes (SMAD4, TP53) that are associated with a higher malignancy risk and/or with cysts featuring an infiltrating adenocarcinoma component.41 

Rosenbaum and colleagues (the same group of Jones et al.41 )42  studied 113 pancreatic cystic lesions with a panel of 39 cancer-related genes. The authors found a total of 119 variants in 67 samples. Most of them were mutations in KRAS (53%; 60 of 113 cases) or GNAS (24%; 27 of 113 cases). Other mutations were found in the following genes: CDKN2A (9%; 10 of 113 cases), TP53 (4%; 5 of 113 cases), and SMAD4 (2%; 2 of 113 cases). Alterations in BRAF, NOTCH1, and PIK3CA were found in only 1 sample each (0.9%; 1 of 113).42  Considering only samples that underwent surgical resection (38; final diagnoses: 8 nonmucinous cysts, 6 IPMNs, and 24 carcinomas), KRAS mutations were found in 75% (18 of 24) of the samples diagnosed as cancer, 16% (1 of 6) of IPMNs, and only in 12.5% (1 of 8) of nonmucinous cystic lesions. GNAS alterations were found more frequently in IPMNs (33.3%; 3 of 6) than in cancers (25.0%; 6 of 24) or nonmucinous cysts (0%; 0 of 8 cases). All the other alterations (TP53, SMAD4, and CDKN2A) were found only in cystic lesions with associated PDAC. Overall, the presence of KRAS mutations had a sensitivity and specificity for cystic mucinous lesions (IPMNs or carcinomas) of 80% and 88%, respectively. In contrast, GNAS mutations had a low sensitivity (27%) but very high specificity (100%) for IPMN. In the study, the combination of NGS with the analysis of CEA in the cyst fluid reached 90% sensitivity and 88% specificity for cystic mucinous lesions.42 

In a retrospective large multicenter study, Springer and colleagues43  showed that the screening of cyst fluid for a panel of genetic markers (gene mutations, loss of heterozygosity, and aneuploidy), in conjunction with the clinical features of the cyst, can be used to fairly accurately classify cystic pancreatic lesions and to identify those cases that require surgical resection. The authors analyzed 11 genes (BRAF, CDKN2A, CTNNB1, GNAS, KRAS, NRAS, PIK3CA, RNF43, SMAD4, TP53, and VHL) known to be mutated in cystic lesions in 130 cyst fluid samples. KRAS was the most frequently mutated gene in IPMNs (78%; 75 of 96 cases) and MCNs (50%; 6 of 12 cases), whereas it was not altered in serous cystadenomas and SPNs. Serous cystadenomas frequently had VHL mutations (42%; 5 of 12 cases), whereas all 10 SPNs harbored mutations in CTNNB1. Overall, the authors observed that when molecular and clinical markers were combined, the sensitivity and specificity in detecting serous cystadenoma increased to 100% and 98%, respectively. For IPMN, the combination of molecular and clinical markers led to higher sensitivity when compared with “composite molecular markers” alone (94% versus 76%), but resulted in a decrease in specificity (84% versus 97%). On the other hand, the use of both molecular and clinical markers decreased sensitivity (90% versus 100%) but increased specificity (97% versus 75%) for the preoperative diagnosis of MCN. For SPN, combining molecular and clinical markers decreased the performance of preoperative assessment: sensitivity decreased to 89% (from 100% using molecular markers alone) and specificity to 92% (100% using molecular markers alone).43 

MicroRNAs are differentially expressed in pancreatic tumors.44  Wang and colleagues45  analyzed by NGS a panel of microRNAs in a cohort of cyst fluid samples from patients with mucinous cysts, IPMNs, and pancreatic cancers. The study showed that multiple microRNAs are differentially abundant in high-grade invasive versus low-grade or benign lesions. Five microRNAs (miR-125, miR-214, miR-26, miR-30, and miR-217) found to be differentially expressed between high-grade and low-grade IPMNs by Wang et al45  were also detected as differentially expressed by Matthaei et al46  in IPMNs analyzed using an array-based real-time PCR method.

Studies that have addressed the NGS analysis of fluid specimens other than cyst fluid are summarized in Table 4. Yu and colleagues47  analyzed the pancreatic juice of patients with PDAC (34 cases), patients with IPMN (57 cases), and controls with normal pancreas or chronic pancreatitis (total of 24 cases). The analysis was performed using NGS for 9 genes (KRAS, GNAS, TP53, SMAD4, CDKN2A, RNF43, TGFBR2, BRAF, and PIK3CA) and digital PCR to evaluate the accuracy of NGS. The pancreatic juice of patients with PDAC had a higher mutational load when compared with that of patients with IPMN (P = .003) and that of control groups (P < .001).47  The most frequently mutated gene in PDAC pancreatic juice was KRAS (91%; 31 of 34 samples). Surprisingly, KRAS was found to be mutated in 42% of controls (10 of 24 cases).47  The second most common mutation in patients with PDAC was TP53 (58%; 20 of 34), which was also detected in patients with IPMN (26%; 15 of 57), but in none of the controls. In addition to KRAS, GNAS and RNF43 were also sometimes mutated in control specimens: 17% (4 of 24 cases) and 4% (1 of 24 cases), respectively. The authors concluded that the identification of mutated KRAS in pancreatic juice is not specific enough to distinguish PDAC from IPMN or nonneoplastic controls. The most specific marker was SMAD4, mutated in only 1 of 80 cases without PDAC (an IPMN of 6 cm with intermediate-grade dysplasia), followed by TP53. The KRAS, SMAD4, and TP53 mutations detected in samples collected before the operation were confirmed in the resection specimens. However, in a few cases, mutations could not be demonstrated in the preoperative pancreatic juice samples, and were only identified in the surgical resections. Thus, the preoperative molecular analysis of pancreatic juice appears to have high positive but low negative predictive value.47  According to the authors, the concentration of TP53 and SMAD4 mutations in pancreatic juice may distinguish patients with PDCA from those with IPMNs and disease controls.47 

Table 4

Next-Generation Sequencing (NGS) Analysis of Pancreatic Lesions Starting From Body Fluid

Next-Generation Sequencing (NGS) Analysis of Pancreatic Lesions Starting From Body Fluid
Next-Generation Sequencing (NGS) Analysis of Pancreatic Lesions Starting From Body Fluid

Zill and colleagues48  analyzed with NGS cell-free DNA from the peripheral blood of 26 patients with advanced pancreatic (n = 18) or biliary (n = 8) carcinomas. KRAS and TP53 were the most frequently mutated genes, but alterations were commonly found also in APC, SMAD4, FBXW7, and BRAF genes. In the study, mutations of KRAS, TP53, APC, FBXW7, and SMAD4 genes had an average sensitivity of 92.3%, specificity of 100%, and diagnostic accuracy of 97.7% when compared with the mutation analysis results from tumor biopsy samples,48  thus demonstrating that NGS analysis of cell-free DNA allows detection of tumor-derived mutations in patients with advanced pancreatobiliary carcinoma.

In spite of the extensive knowledge about the molecular alterations of pancreatic tumors accumulated in the recent past, the mortality rate of PDAC patients remains very high. As stated by the current European Society for Medical Oncology guidelines,49  to date there are not targetable molecules for personalized patient treatment. Of the several markers investigated, KRAS remains the one most commonly used for single-gene testing, although its use is greatly limited by the identification of mutated KRAS in about 10% of chronic pancreatitis and/or low-grade pancreatobiliary epithelial cell dysplasia (in some studies frequencies >10% have been reported).5053  Thus, the Papanicolaou Society of Cytopathology guidelines do not support KRAS testing of solid pancreatic masses and bile duct strictures as a useful single-gene ancillary test. The same guidelines report that “a number of gene mutations (KRAS, GNAS, VHL, RNF43 and CTNNB1) may be of aid in the identification of specific cystic neoplasms.” 50 Given this context, NGS may be instrumental for the preoperative molecular diagnosis of pancreatic lesions, because it offers the opportunity of screening simultaneously a wide number of mutations while using small amounts of DNA. It is becoming clear that the use of wide gene panels increases clinical sensitivity and specificity, minimizing the risk of false-positive results. High analytical sensitivity and positive predictive value can prevent repeat biopsies, and thus improve preoperative diagnosis and preoperative patient risk stratification and management, while reducing costs. On the other hand, NGS requires expensive instrumentation and specialized expertise, and its application to the preoperative diagnosis needs a robust validation in large multicenter series of paired preoperative and surgical samples. Further studies, with careful evaluation of costs versus benefits, will be necessary before the full implementation of NGS in clinical practice.

This work was supported in part by Italian Government-Ministero Della Salute grant RF-2011-02350857 to G.T.

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Author notes

The authors have no relevant financial interest in the products or companies described in this article.

Competing Interests

Presented in part at the V Molecular Cytopathology: Focus on Next Generation Sequencing in Cytopathology meeting; October 18, 2016; Napoli, Italy.