Printer Friendly

The Role of Next-Generation Sequencing in the Cytologic Diagnosis of Pancreatic Lesions.

Pancreatic 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 described (5): 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. (9-14)

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. (18-21)

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.

NGS OF EUS-FNA SAMPLES OF SOLID PANCREATIC LESIONS

The studies that addressed NGS analysis of EUS-FNA samples of solid lesions are summarized in Table 2. In 2014, De Biase et al (13) 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)

Kameta et al (23) 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 colleagues (24) 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), CDKN2AJB (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 colleagues (25) 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 mutation (28) 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 colleagues (29) 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 colleagues (30) 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 authors (30) 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 colleagues (33) 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 ARIDI (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 [beta]-catenin. Kubota et al (34) 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.

NGS OF EUS-FNA SAMPLES OF PANCREATIC CYST FLUID

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)

Al-Haddad and colleagues (36) 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 al (39) 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 al (41) 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 colleagues (43) 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 colleagues (45) 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 al (45) were also detected as differentially expressed by Matthaei et al (46) in IPMNs analyzed using an array-based real-time PCR method.

NEXT-GENERATION ANALYSIS OF OTHER FLUID BIOLOGICAL SPECIMENS FROM PATIENTS WITH PANCREATIC TUMORS

Studies that have addressed the NGS analysis of fluid specimens other than cyst fluid are summarized in Table 4. Yu and colleagues (47) 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)

Zill and colleagues (48) 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.

CONCLUSIONS AND FUTURE PERSPECTIVE

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). (50-53) 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.

References

(1.) Ryan DP, Hong TS, Bardeesy N. Pancreatic adenocarcinoma. N Engl J Med. 2014;371 (11):1039-1049.

(2.) Hong SK, Loren DE, Rogart JN, et al. Targeted cyst wall puncture and aspiration during EUS-FNA increases the diagnostic yield of premalignant and malignant pancreatic cysts. Gastrointest Endosc. 2012;75(4):775-782.

(3.) Dumonceau JM, Polkowski M, Larghi A, et al. Indications, results, and clinical impact of endoscopic ultrasound (EUS)-guided sampling in gastroenterology: European Society of Gastrointestinal Endoscopy (ESCE) clinical guideline. Endoscopy. 2011;43(10):897-912.

(4.) Jenssen C, Hocke M, Fusaroli P, et al. EFSUMB Guidelines on Interventional Ultrasound (INVUS), part IV--EUS-guided interventions: general aspects and EUS-guided sampling (long version). Ultraschall Med. 2016;37(2): E33-E76.

(5.) Hawass NE. Comparing the sensitivities and specificities of two diagnostic procedures performed on the same group of patients. Br J Radiol. 1997;70(832): 360-366.

(6.) Varadarajulu S, Fockens P, Hawes RH. Best practices in endoscopic ultrasound-guided fine-needle aspiration. Clin Gastroenterol Hepatol. 2012; 10(7):697-703.

(7.) Eser S, Schnieke A, Schneider G, Saur D. Oncogenic KRAS signalling in pancreatic cancer. Br J Cancer. 2014;111(5):817-822.

(8.) Iacobuzio-Donahue CA. Genetic evolution of pancreatic cancer: lessons learnt from the pancreatic cancer genome sequencing project. Gut. 2012;61(7): 1085-1094.

(9.) Khalid A, Nodit L, Zahid M, et al. Endoscopic ultrasound fine needle aspirate DNA analysis to differentiate malignant and benign pancreatic masses. Am J Gastroenterol. 2006;101(11):2493-2500.

(10.) Tada M, Komatsu Y, Kawabe T, et al. Quantitative analysis of K-ras gene mutation in pancreatic tissue obtained by endoscopic ultrasonography-guided fine needle aspiration: clinical utility for diagnosis of pancreatic tumor. Am J Gastroenterol. 2002;97(9):2263-2270.

(11.) Khalid A, Dewitt J, Ohori NP, et al. EUS-FNA mutational analysis in differentiating autoimmune pancreatitis and pancreatic cancer. Pancreatology. 2011;11(5):482-486.

(12.) Goggins M. Identifying molecular markers for the early detection of pancreatic neoplasia. Semin Oncol. 2007;34(4):303-310.

(13.) de Biase D, Visani M, Baccarini P, et al. Next generation sequencing improves the accuracy of KRAS mutation analysis in endoscopic ultrasound fine needle aspiration pancreatic lesions. PLoS One. 2014;9(2):e87651.

(14.) Dillon DA, Johnson CC, Topazian MD, Tallini G, Rimm DL, Costa JC. The utility of Ki-ras mutation analysis in the cytologic diagnosis of pancreatobiliary neoplasma. Cancer J. 2000;6(5):294-301.

(15.) Jones S, Zhang X, Parsons DW, et al. Core signaling pathways in human pancreatic cancers revealed by global genomic analyses. Science. 2008; 321(5897):1801-1806.

(16.) Di Marco M, Astolfi A, Grassi E, et al. Characterization of pancreatic ductal adenocarcinoma using whole transcriptome sequencing and copy number analysis by single-nucleotide polymorphism array. Mol Med Rep. 2015;12(5): 7479-7484.

(17.) Liang WS, Craig DW, Carpten J, et al. Genome-wide characterization of pancreatic adenocarcinoma patients using next generation sequencing. PLoS One. 2012;7(10):e43192.

(18.) Forbes SA, Beare D, Boutselakis H, et al. COSMIC: somatic cancer genetics at high-resolution. Nucleic Acids Res. 2017;45(D1):D777-D783.

(19.) La Rosa S, Sessa F, Capella C. Acinar cell carcinoma of the pancreas: overview of clinicopathologic features and insights into the molecular pathology. Front Med (Lausanne). 2015;2:41.

(20.) Chen M, Van Ness M, Guo Y, Gregg J. Molecular pathology of pancreatic neuroendocrine tumors. J Gastrointest Oncol. 2012;3(3):182-188.

(21.) Reid MD, Choi H, Balci S, Akkas G, Adsay V. Serous cystic neoplasms of the pancreas: clinicopathologic and molecular characteristics. Semin Diagn Pathol. 2014;31(6):475-483.

(22.) de Biase D, de Luca C, Gragnano G, et al. Fully automated PCR detection of KRAS mutations on pancreatic endoscopic ultrasound fine-needle aspirates [published online ahead of print April 27, 2016]. J Clin Pathol. doi:10.1136/ jclinpath-2016-203696.

(23.) Kameta E, Sugimori K, Kaneko T, et al. Diagnosis of pancreatic lesions collected by endoscopic ultrasound-guided fine-needle aspiration using next- generation sequencing. Oncol Lett. 2016;12(5):3875-3881.

(24.) Young G, Wang K, He J, et al. Clinical next-generation sequencing successfully applied to fine-needle aspirations of pulmonary and pancreatic neoplasms. Cancer Cytopathol. 2013;121(12):688-694.

(25.) Gleeson FC, Kerr SE, Kipp BR, et al. Targeted next generation sequencing of endoscopic ultrasound acquired cytology from ampullary and pancreatic adenocarcinoma has the potential to aid patient stratification for optimal therapy selection. Oncotarget. 2016;7(34):54526-54536.

(26.) Laghi L, Orbetegli O, Bianchi P, et al. Common occurrence of multiple KRAS mutations in pancreatic cancers with associated precursor lesions and in biliary cancers. Oncogene. 2002;21(27):4301-4306.

(27.) Visani M, de Biase D, Baccarini P, et al. Multiple KRAS mutations in pancreatic adenocarcinoma: molecular features of neoplastic clones indicate the selection of divergent populations of tumor cells. Int J Surg Pathol. 2013;21(6): 546-552.

(28.) de Biase D, Cesari V, Visani M, et al. High-sensitivity BRAF mutation analysis: BRAF V600E is acquired early during tumor development but is heterogeneously distributed in a subset of papillary thyroid carcinomas. J Clin Endocrinol Metab. 2014;99(8):E1530-E1538.

(29.) Sibinga Mulder BG, Mieog JS, Handgraaf HJ, et al. Targeted next-generation sequencing of FNA-derived DNA in pancreatic cancer. J Clin Pathol. 2017;70(2):174-178.

(30.) Dudley JC, Zheng Z, McDonald T, et al. Next-generation sequencing and fluorescence in situ hybridization have comparable performance characteristics in the analysis of pancreaticobiliary brushings for malignancy. J Mol Diagn. 2016; 18(1):124-130.

(31.) Arvanitakis M, Van Laethem JL, Parma J, De Maertelaer V, Delhaye M, Deviere J. Predictive factors for pancreatic cancer in patients with chronic pancreatitis in association with K-ras gene mutation. Endoscopy. 2004;36(6): 535-542.

(32.) Kamisawa T, Takuma K, Tabata T, Egawa N, Yamaguchi T. Long-term follow-up of chronic pancreatitis patients with K-ras mutation in the pancreatic juice. Hepatogastroenterology. 2011;58(105):174-176.

(33.) Valero V 3rd, Saunders TJ, He J, et al. Reliable detection of somatic mutations in fine needle aspirates of pancreatic cancer with next-generation sequencing: implications for surgical management. Ann Surg. 2016;263(1):153-161.

(34.) Kubota Y, Kawakami H, NatsuizakaM, et al. CTNNB1 mutational analysis of solid-pseudopapillary neoplasms of the pancreas using endoscopic ultrasound- guided fine-needle aspiration and next-generation deep sequencing. J Gastroenterol. 2015;50(2):203-210.

(35.) Basturk O, Coban I, Adsay NV. Pancreatic cysts: pathologic classification, differential diagnosis, and clinical implications. Arch Pathol Lab Med. 2009; 133(3):423-438.

(36.) Al-Haddad M, DeWitt J, Sherman S, et al. Performance characteristics of molecular (DNA) analysis for the diagnosis of mucinous pancreatic cysts. Gastrointest Endosc. 2014;79(1):79-87.

(37.) van der Waaij LA, van Dullemen HM, Porte RJ. Cyst fluid analysis in the differential diagnosis of pancreatic cystic lesions: a pooled analysis. Gastrointest Endosc. 2005;62(3):383-389.

(38.) Oh HC, Kang H, Brugge WR. Cyst fluid amylase and CEA levels in the differential diagnosis of pancreatic cysts: a single-center experience with histologically proven cysts. Dig Dis Sci. 2014;59(12):3111-3116.

(39.) Amato E, Molin MD, Mafficini A, et al. Targeted next-generation sequencing of cancer genes dissects the molecular profiles of intraductal papillary neoplasms of the pancreas. J Pathol. 2014;233(3):217-227.

(40.) Singhi AD, Nikiforova MN, Fasanella KE, et al. Preoperative GNAS and KRAS testing in the diagnosis of pancreatic mucinous cysts. Clin Cancer Res. 2014;20(16):4381-4389.

(41.) Jones M, Zheng Z, Wang J, et al. Impact of next-generation sequencing on the clinical diagnosis of pancreatic cysts. Gastrointest Endosc. 2016;83(1):140-148.

(42.) Rosenbaum MW, Jones M, Dudley JC, Le LP, Iafrate AJ, Pitman MB. Next- generation sequencing adds value to the preoperative diagnosis of pancreatic cysts. Cancer. 2017;125(1):41-47.

(43.) Springer S, Wang Y, Dal Molin M, et al. A combination of molecular markers and clinical features improve the classification of pancreatic cysts. Gastroenterology. 2015;149(6):1501-1510.

(44.) Muller S, Raulefs S, Bruns P, et al. Next-generation sequencing reveals novel differentially regulated mRNAs, lncRNAs, miRNAs, sdRNAs and a piRNA in pancreatic cancer. Mol Cancer. 2015;14:94.

(45.) Wang J, Paris PL, Chen J, et al. Next generation sequencing of pancreatic cyst fluid microRNAs from low grade-benign and high grade-invasive lesions. Cancer Lett. 2015;356(2, pt B):404-409.

(46.) Matthaei H, Wylie D, Lloyd MB, et al. miRNA biomarkers in cyst fluid augment the diagnosis and management of pancreatic cysts. Clin Cancer Res. 2012;18(17):4713-4724.

(47.) Yu J, Sadakari Y, Shindo K, et al. Digital next-generation sequencing identifies low-abundance mutations in pancreatic juice samples collected from the duodenum of patients with pancreatic cancer and intraductal papillary mucinous neoplasms. Gut. 2017;66(9):1677-1687.

(48.) Zill OA, Greene C, Sebisanovic D, et al. Cell-free DNA next-generation sequencing in pancreatobiliary carcinomas. Cancer Discov. 2015;5(10):1040-1048.

(49.) Ducreux M, Cuhna AS, Caramella C, et al. Cancer of the pancreas: ESMO clinical practice guidelines for diagnosis, treatment and follow-up. Ann Oncol 2015;26(suppl 5):v56-v68.

(50.) Layfield LJ, Ehya H, Filie AC, et al. Utilization of ancillary studies in the cytologic diagnosis of biliary and pancreatic lesions: the Papanicolaou Society of Cytopathology guidelines for pancreatobiliary cytology. Diagn Cytopathol. 2014; 42(4):351-362.

(51.) Luttges J, Reinecke-Luthge A, Mollmann B, et al. Duct changes and K-ras mutations in the disease-free pancreas: analysis of type, age relation and spatial distribution. Virchows Arch. 1999;435(5):461-468.

(52.) Lohr M, Kloppel G, Maisonneuve P, Lowenfels AB, Luttges J. Frequency of K-ras mutations in pancreatic intraductal neoplasias associated with pancreatic ductal adenocarcinoma and chronic pancreatitis: a meta-analysis. Neoplasia. 2005;7(1):17-23.

(53.) Lohr M, Maisonneuve P, Lowenfels AB. K-ras mutations and benign pancreatic disease. Int J Pancreatol. 2000;27(2):93-103.

Accepted for publication October 4, 2017.

From the Department of Pharmacy and Biotechnology (Dipartimento di Farmacia e Biotecnologie)--Molecular Diagnostic Unit, Azienda USL di Bologna, University of Bologna, Bologna, Italy (Dr de Biase and Ms Pession); the Department of Medicine (Dipartimento di Medicina Specialistica, Diagnostica e Sperimentale)--Molecular Diagnostic Unit, Azienda USL di Bologna, University of Bologna School of Medicine, Bologna, Italy (Drs Visani and Tallini and Ms Acquaviva); the Unit of Anatomic Pathology, Azienda USL-Maggiore Hospital, Bologna, Italy (Dr Fornelli); and the Units of Surgery (Dr Masetti) and Gastroenterology and Digestive Endoscopy (Dr Fabbri), Azienda USL Bologna Bellaria-Maggiore Hospitals, Bologna, Italy.

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

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

Reprints: Giovanni Tallini, MD, Department of Medicine (Dipartimento di Medicina Specialistica, Diagnostica e Sperimentale), University of Bologna School of Medicine, Anatomic Pathology Unit, Ospedale Bellaria, Via Altura 3, 40139 Bologna, Italy (email: giovanni.tallini@unibo.it).
Table 1. Main Genetic Alterations in
Pancreatic Tumors

Pancreatic
Tumor         Gene Altered   Frequency, %

PDAC          KRAS                95
              TP53              35-40
              CDKN2A/p16        95-100
              SMAD4             45-60
IPMN          KRAS              45-50
              GNAS              35-50
MCN           KRAS              20-50
              GNAS              25-50
PanIN         KRAS                70
              TP53                20
              GNAS                5
SPN           CTNNB1            90-100
PAAC          APC               50-60
              CTNNB1             5-10
              TP53              30-40
SCA           VHL               40-50 (a)
P-NET         MEN1              10-30 (b)

Abbreviations: IPMN, intraductal papillary mucinous neoplasm; MCN,
mucinous cystic neoplasm; PAAC, pancreatic acinic adenocarcinoma;
PanIN, pancreatic intraepithelial neoplasia; PDAC, pancreatic ductal
adenocarcinoma; P-NET, pancreatic neuroendocrine tumor; SCA,
serous cystadenoma; SPN, solid pseudopapillary neoplasm.

(a) Somatic alterations (mutation or loss of heterozygosity) in sporadic
tumors; germline mutations are present in patients with the von
Hippel-Lindau syndrome.

(b) Somatic mutations in sporadic tumors; germline mutations for MEN1,
VHL, NF-1, or TSC1/2 are present in P-NET that develop in patients
with inherited genetic neuroendocrine syndromes. The percentages
quoted are estimated from the literature quoted in the paper.

Table 2. Next-Generation Sequencing Analysis of Solid Pancreatic Tumors
Starting From Fine-Needle Aspiration (FNA) Material

                     Type of Tumor
Source, y                (No.)             Genes Targeted

Young et al,      PDAC (18), P-NET      Panel of 287 cancer-
  (24) 2013         (1), PC NOS (2)       related genes
                                          and 19 genes
                                          frequently
                                          rearranged
                                          in cancer

de Biase,         PDAC (20),            KRAS
  2014 (13)         inoperable
                    pancreatic tumors
                    (6), P-NET (5)

Di Marco et       PDAC (16)             Whole-exome
  al, (16) 2015                           sequencing

Kubota et         SPN (7), PDAC (16),   CTNNB1
  al, (34) 2015     P-NET (11)

Dudley et al,     Bile and main         Custom panel (39
  (30) 2016         pancreatic duct       cancer-related
                    samples:              genes)
                    neoplastic (31),
                    nonneoplastic
                    (43)

Gleeson et al,    PDAC (22), AA (4)     Human
  (25) 2016                               Comprehensive
                                          Cancer GeneRead
                                          DNAseq Targeted
                                          Panel V2 (panel
                                          of 160 cancer
                                          genes)

Kameta et al,     PDAC (27)             AmpliSeq Cancer
  (23) 2016                               Panel v2 (50
                                          genes)

Sibinga Mulder    PDAC (1)              AmpliSeq Cancer
  et al,                                  Panel V2 (50
  (29) 2017                               genes)

Valero et al,     Unresectable PDAC     Ion Ampliseq
  (33) 2016       (19)                    Comprehensive
                                          Cancer Panel (409
                                          cancer-related
                                          genes)

                                                       More Frequent
Source, y             Platform        Material        Alterations (%)

Young et al,      HiSeq 2000         FNA          KRAS (76.2), TP53
  (24) 2013         (Illumina) (a)                  (71.4), CDKN2A/2B
                                                    (38.1), SMAD4
                                                    (19.0), PTEN
                                                    (14.3)

de Biase,         454 GS-            EUS-guided   PDAC and inoperable
  2014 (13)         Junior             FNA          pancreatic tumors:
                    (Roche) (b)                     KRAS (93.5)
                                                    P-NET: KRAS (0)

Di Marco et       HiScanSQ           EUS-FNB      KRAS (93.7), TP53
  al, (16) 2015     (Illumina)                      (56.0), CDKN2A
                                                    (50.0)

Kubota et         Ion PGM            EUS-FNA      SPN: CTNNB1 (100)
  al, (34) 2015     (Thermo                         P-NET: CTNNB1 (9)
                    Fisher) (c)                     PDAC: CTNNB1 (0)

Dudley et al,     MiSeq              Brushing     Neoplastic: KRAS
  (30) 2016         (Illumina)                      (64.5), TP53
                                                    (45.2), SMAD4
                                                    (19.4), CDKN2A
                                                    (12.9) Nonneo-
                                                    plastic: KRAS (2.3)

Gleeson et al,    HiSeq 2000         EUS-FNA      KRAS (93.1),d TP53
  (25) 2016         (Illumina)                      (72.4), (d) SMAD4
                                                    (31.0), (d) GNAS
                                                    (10.3) (d)

Kameta et al,     Ion PGM            EUS-FNA      KRAS (96.0), TP53
  (23) 2016         (Thermo                         (44.0), CDKN2A
                    Fisher)                         (15.0), SMAD4
                                                    (11.0)

Sibinga Mulder    Ion PGM            EUS-FNA      KRAS, TP53, CDKN2A
  et al,          (Thermo
  (29) 2017       Fisher)

Valero et al,     Ion PGM            FNA          KRAS (73.7), TP53
  (33) 2016       (Thermo                           (47.4), SMAD4
                  Fisher)                           (31.6), ARID1
                                                    (15.8)

Abbreviations: AA, ampullary adenocarcinoma; EUS, endoscopic
ultrasound-guided; FNB, fine-needle biopsy; PC NOS, pancreatic
carcinomas not otherwise specified; PDAC, pancreatic ductal
adenocarcinoma; P-NET, pancreatic neuroendocrine tumor; SPN, solid
pseudopapillary neoplasm.

(a) Illumina Inc, San Diego, California.

(b) Roche Diagnostics, Mannheim, Germany.

(c) Thermo Fisher Scientific, Waltham, Massachusetts.

(d) It is not possible to discriminate the mutation prevalence of
solid versus cystic lesions: 3 intraductal papillary mucinous
neoplasms are included in the study (see Table 3).

Table 3. Next-Generation Sequencing Analysis of Cystic Pancreatic
Lesions Starting From Fine-Needle Aspiration (FNA) Material

                     Type of Tumor
Source, y                (No.)             Genes Targeted

Young et al,       MCN (2, both         Panel of 287 cancer-
  (24) 2013          adenocarcinomas)     related genes and
                                          19 genes
                                          frequently
                                          rearranged in
                                          cancer
Amato et al,       IPMN (7)             AmpliSeq Cancer
  (39) 2014                               Panel v2 (50
                                          genes)
Wang et al,        IPMN, MCN, PC        Noncoding RNA
  (45) 2015          NOS (17 total)

de Biase et al,    Cyst NOS (26)        KRAS
  (13) 2014

Springer et al,    IPMN (96), MCN       Panel of 11 custom
  (43) 2015          (12), SCA (12),      cancer-related
                     SPN (10)             genes

Gleeson et al,     IPMN (3)             Human
  (41) 2016                               Comprehensive
                                          Cancer GeneRead
                                          DNAseq Targeted
                                          Panel V2 (160
                                          cancer genes
                                          panel)

Jones et al,       Cyst NOS (92)        Custom panel (39
  (15) 2016                               cancer-related
                                          genes)

Rosenbaum et       Cyst NOS (113)       Custom panel (39
  al, (42) 2017)                          cancer-related
                                          genes)

                                                    More Frequent
Source, y             Platform        Material     Alterations (%)

Young et al,       HiSeq 2000        FNA          KRAS (100.0),
  (24) 2013          (Illumina)                     TP53 (100.0)
                     (a)

Amato et al,       Ion PGM (Thermo   Cyst fluid   GNAS, KRAS,
  (39) 2014          Fisher) (b)                    TP53c

Wang et al,        SoliD (Thermo     Cyst fluid   15 miRNAs
  (45) 2015          Fisher)                        differently
                                                    expressed
                                                    between low-
                                                    grade and high-
                                                    grade lesions

de Biase et al,    454 GS-Junior     EUS-FNA      KRAS: 38.5 (83.3
  (13) 2014          (Roche) (d)                    in IPMN)

Springer et al,    MiSeq             Cyst fluid   IPMN: KRAS
  (43) 2015          (Illumina)                     (78.0), GNAS
                                                    (58.0), RNF43
                                                    (38.0)
                                                    MCN: KRAS
                                                      (50.0)
                                                    SCA: VHL
                                                      (42.0)
                                                    SPN: CTNNB1
                                                      (100.0),
                                                      PIK3CA
                                                      (20.0)

Gleeson et al,     HiSeq 2000        EUS-FNA          NA (e)
  (41) 2016          (Illumina)

Jones et al,       Anchored          EUS-FNA      KRAS (47.0),
  (15) 2016          multiplex                      GNAS (24.0)
                     PCR NGS
                     platform

Rosenbaum et       Anchored          Cyst fluid   Cancer: KRAS
  al, (42) 2017)     multiplex                      (75.0), GNAS
                     PCR NGS                        (25.0), TP53
                     platform                       (16.7), CDKN2A
                                                    (33.3) (f)
                                                    IPMN: KRAS
                                                    (100.0),
                                                    GNAS (33.0) (f)
                                                  Nonmucinous:
                                                    KRAS (12.5) (f)

Abbreviations: Cyst NOS, cystic lesions not otherwise specified; EUS,
endoscopic ultrasound-guided; IPMN, intraductal papillary mucinous
neoplasm; MCN, mucinous cystic neoplasm; PC NOS, pancreatic carcinomas
not otherwise specified; SCA, serous cystadenoma; SPN, solid
pseudopapillary neoplasm.

(a) Illumina Inc, San Diego, California.

(b) Thermo Fisher Scientific, Waltham, Massachusetts.

(c) Data about prevalence of mutations in the 7 IPMN cyst fluids are
not available.

(d) Roche Diagnostics, Mannheim, Germany.

(e) Data about prevalence of mutations in the IPMN cyst fluids are not
available (see Table 2).

(f) Calculated on the 24 cysts with a final diagnosis in the series;
the studies by Jones et al (41) and Rosenbaum et al (42) are from the
same group.

Table 4. Next-Generation Sequencing (NGS) Analysis of Pancreatic
Lesions Starting From Body Fluid

Source, y       Type of Tumor (No)     Genes Targeted

Zill et al,     Advanced             Panel of 54 custom
(48) 2015         pancreatobiliary     cancer-related
                  carcinoma (26)       genes

Yu et al,       PDAC (34)            Panel of 9 custom
(47) 2017 (b)                          cancer-related
                                       genes

                                                 More Frequent
Source, y           Platform        Material    Alterations, %

Zill et al,     HiSeq 2500         Blood        KRAS (53.8),
(48) 2015         (Illumina) (a)     (cfDNA)      TP53 (38.5)

Yu et al,       NGS (Thermo        Pancreatic   KRAS (74.0),
(47) 2017 (b)     Fisher) (c)        juice        TP53 (59.0),
                                                  GNAS (38.0),
                                                  RNF43 (24.0),
                                                  SMAD4 (15.0)

Abbreviations: cfDNA, cell-free DNA; PDAC, pancreatic ductal
adenocarcinoma.

(a) Illumina Inc, San Diego, California.

(b) Results obtained using both NGS and digital polymerase
chain reaction.

(c) Thermo Fisher Scientific, Waltham, Massachusetts.
COPYRIGHT 2018 College of American Pathologists
No portion of this article can be reproduced without the express written permission from the copyright holder.
Copyright 2018 Gale, Cengage Learning. All rights reserved.

Article Details
Printer friendly Cite/link Email Feedback
Author:de Biase, Dario; Visani, Michela; Acquaviva, Giorgia; Fornelli, Adele; Masetti, Michele; Fabbri, Car
Publication:Archives of Pathology & Laboratory Medicine
Article Type:Report
Geographic Code:4EUIT
Date:Apr 1, 2018
Words:6652
Previous Article:Update on Molecular Testing for Cytologically Indeterminate Thyroid Nodules.
Next Article:Acquired Resistance to Tyrosine Kinase Inhibitors in Non-Small Cell Lung Cancers: The Role of Next-Generation Sequencing on Endobronchial...
Topics:

Terms of use | Privacy policy | Copyright © 2021 Farlex, Inc. | Feedback | For webmasters |