Pancreatic ductal adenocarcinoma (PDAC) is one of the

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1 The Role of Next-Genertion Sequencing in the Cytologic Dignosis of Pncretic Lesions Drio de Bise, PhD; Michel Visni, PhD; Giorgi Acquviv, MSc; Adele Fornelli, MD; Michele Msetti, MD; Crlo Fri, MD; Annlis Pession, BSc; Giovnni Tllini, MD Context. Integrtion of the nlysis of genetic mrkers with endoscopic ultrsound guided fine-needle spirtion nd cytologic evlution hs incresed the ccurcy of the preopertive dignosis of pncretic lesions. The ppliction of high-throughput gene pnel nlysis using nextgenertion sequencing pltforms is now offering gret opportunity for further improvements. Ojective. To review the ppliction of next-genertion sequencing to the preopertive dignosis of pncretic lesions. Dt Sources. For dt cquisition, PuMed serch using the terms next-genertion sequencing, pncres, pncretic lesions, pncretic tumors, nd EUS-FNA ws performed covering the yers Conclusions. KRAS remins the gene most widely studied for preopertive single-gene tests. Next-genertion sequencing relily llows nlysis of multiple gene mrkers strting from limited mounts of DNA. The study of multigene pnels hs ecome very ttrctive option for the mngement nd preopertive risk strtifiction of ptients with pncretic cncer. (Arch Pthol L Med. 2018;142: ; doi: / rp ra) Pncretic ductl denocrcinom (PDAC) is one of the most ggressive types of cncer in humns nd is usully dignosed t n dvnced stge. 1 The mngement of ptients with pncretic mss (tumor or cysts) is still chllenge. Endoscopic ultrsound (EUS) guided fine-needle spirtion (FNA) hs gretly improved preopertive dignosis, 2 with smple dequcy (ie, the cquisition of dignostic mteril) currently rnging from 65% to 96%. 3,4 Although EUS-FNA plus cytologic evlution shows high clinicl sensitivity (clculted ccording to recommendtions previously descried 5 : true-positive/[true-positive þ flse-negtive]) nd specificity (true-negtive/[true-negtive Accepted for puliction Octoer 4, From the Deprtment of Phrmcy nd Biotechnology (Diprtimento di Frmci e Biotecnologie) Moleculr Dignostic Unit, Aziend USL di Bologn, University of Bologn, Bologn, Itly (Dr de Bise nd Ms Pession); the Deprtment of Medicine (Diprtimento di Medicin Specilistic, Dignostic e Sperimentle) Moleculr Dignostic Unit, Aziend USL di Bologn, University of Bologn School of Medicine, Bologn, Itly (Drs Visni nd Tllini nd Ms Acquviv); the Unit of Antomic Pthology, Aziend USL- Mggiore Hospitl, Bologn, Itly (Dr Fornelli); nd the Units of Surgery (Dr Msetti) nd Gstroenterology nd Digestive Endoscopy (Dr Fri), Aziend USL Bologn Bellri-Mggiore Hospitls, Bologn, Itly. The uthors hve no relevnt finncil interest in the products or compnies descried in this rticle. Presented in prt t the V Moleculr Cytopthology: Focus on Next Genertion Sequencing in Cytopthology meeting; Octoer 18, 2016; Npoli, Itly. Reprints: Giovnni Tllini, MD, Deprtment of Medicine (Diprtimento di Medicin Specilistic, Dignostic e Sperimentle), University of Bologn School of Medicine, Antomic Pthology Unit, Ospedle Bellri, Vi Altur 3, Bologn, Itly (emil: giovnni.tllini@unio.it). þ flse-positive]), in suset of cses the preopertive dignosis remins inconclusive ecuse of indequte/ insufficient mteril or limited cellulrity, leding to typicl/suspicious cytopthologic dignoses. 6 In most cses, PDAC is initited y oncogenic mutnt KRAS, which hs een shown to drive pncretic neoplsi. 7,8 In the pst 2 decdes severl studies hve shown how the nlysis of KRAS muttions of pncretic lesions improves dignostic ccurcy nd is prticulrly useful in those cses where EUS-FNA cytology is inconclusive Next-genertion sequencing (NGS) hs een instrumentl for the understnding of the pncretic cncer genome. Whole-exome sequencing of genes hs shown tht ny given PDAC contins n verge of 63 genomic ltertions, most of which re point muttions. 15 Highthroughput moleculr nlysis hs demonstrted tht in ddition to KRAS muttion, inctivtions of TP53, SMAD4, nd CDKN2A/p16 re the pivotl moleculr ltertions tht define the development nd progression of PDAC. 16,17 This finding, nd the vriety of moleculr ltertions found in different types of pncretic tumors (summrized in Tle 1), such s CTNNB1 muttions in solid pseudoppillry neoplsm (SPN) or GNAS muttions in intrductl ppillry mucinous neoplsm (IPMN), underscore the need for the nlysis of multiple iomrkers The introduction of NGS to moleculr dignostics hs llowed performnce of moleculr nlysis of multiple genes in limited smples, opening new venues to the study of preopertive specimens. Preopertive cytologic smers (ie, mteril otined during FNA or iliry rushings smered on slide) nd fine-needle iopsies (ie, smll smples otined using 22-guge coring needle tht provides cylindricl specimens for the exmintion of tumor rchitecture) re chrcterized y smll quntity of dignostic 458 Arch Pthol L Med Vol 142, April 2018 NGS of Pncretic EUS-FNA de Bise et l

2 Tle 1. Min Genetic Altertions in Pncretic Tumors Pncretic Tumor Gene Altered Frequency, % PDAC KRAS 95 TP CDKN2A/p SMAD IPMN KRAS GNAS MCN KRAS GNAS PnIN KRAS 70 TP53 20 GNAS 5 SPN CTNNB PAAC APC CTNNB TP SCA VHL P-NET MEN Arevitions: IPMN, intrductl ppillry mucinous neoplsm; MCN, mucinous cystic neoplsm; PAAC, pncretic cinic denocrcinom; PnIN, pncretic intrepithelil neoplsi; PDAC, pncretic ductl denocrcinom; P-NET, pncretic neuroendocrine tumor; SCA, serous cystdenom; SPN, solid pseudoppillry neoplsm. Somtic ltertions (muttion or loss of heterozygosity) in spordic tumors; germline muttions re present in ptients with the von Hippel Lindu syndrome. Somtic muttions in spordic tumors; germline muttions for MEN1, VHL, NF-1, or TSC1/2 re present in P-NET tht develop in ptients with inherited genetic neuroendocrine syndromes. The percentges quoted re estimted from the literture quoted in the pper. mteril, nd re often composed of heterogeneous cell popultions. For this reson, it is crucil to use moleculr tests tht offer high nlyticl sensitivity to detect smll proportions of mutted cells. Next-genertion sequencing comines high nlyticl sensitivity with multiple gene nlysis nd thus represents very ttrctive option. The scope of this review is to provide n updted survey of the studies tht hve used NGS for the preopertive moleculr workup of pncretic lesions. To identify the pertinent references, PuMed serch using the terms nextgenertion sequencing, pncres, pncretic lesions, pncretic tumors, nd EUS-FNA ws performed covering the yers Our review ddresses studies tht nlyzed (1) solid pncretic lesions, (2) pncretic cyst fluid, or (3) other fluid specimens from ptients with pncretic tumors. NGS OF EUS-FNA SAMPLES OF SOLID PANCREATIC LESIONS The studies tht ddressed NGS nlysis of EUS-FNA smples of solid lesions re summrized in Tle 2. In 2014, De Bise et l 13 demonstrted tht NGS is more sensitive nd specific thn conventionl muttion nlysis for the preopertive identifiction of pncretic lesions with mlignnt potentil. The uthors oserved tht using NGS to nlyze KRAS muttions in pncretic FNA specimens llows clinicl sensitivity to increse up to ~74%, vlue fr superior to tht otined y llele-specific rel-time polymerse chin rection (PCR) (52.8%) or y Snger sequencing (42.1%), while mintining clinicl specificity t 100%. The nlyticl sensitivity ws higher if the nlysis ws performed strting from cells scrped from the smer preprtions used for routine cytologic dignosis, s opposed to FNA mteril directly sumitted y the endoscopist for moleculr dignosis. 13 Trgeted KRAS prllel sequencing hd higher clinicl sensitivity when compred with commercilly ville KRAS muttion specific dignostic kits. 22 Kmet et l 23 nlyzed pnel of 50 most commonly mutted oncogenes (Ampliseq Cncer Hotspot Pnel v2.0, designed to mplify 207 mplicons covering ~2790 muttions; Thermo Fisher Scientific, Wlthm, Msschusetts) in cohort of 38 ptients with pncretic disese, 27 of whom were dignosed with PDAC. The sequencing ws performed on DNA extrcted from EUS-FNA mteril nd showed tht KRAS ws the most frequently mutted gene (96%; 26 of 27 PDACs). TP53 muttions were detected in 44% of the smples (12 of 27) nd oth SMAD4 nd CDKN2A in 11% (3 of 27). Intriguingly, 2 muttions detected y NGS were not detected y the rel-time PCR ssy used y the uthors for comprison (presumly ecuse the muttion-specific rel-time PCR ssy ws not designed to detect them). The uthors lso nlyzed smples from metsttic tumors of unknown origin to investigte if the pnel might e of help to identify the primry lesion, ut the results were limited y the lck of specificity of KRAS muttions, which were commonly present not only in PDAC ut lso in denocrcinoms of lung, colon, nd stomch. They concluded tht their ssy could e used for the preopertive dignosis of pncretic EUS-FNA, even if 50-gene pnel might not e sufficient to fully profile the moleculr lndscpe of PDAC. Young nd collegues 24 tested lrge multigene pnel (287 genes, covering totl of 4561 exons nd 47 introns of 19 genes frequently rerrnged in cncer) on 23 pncretic FNAs. The cohort consisted of 17 PDACs, 3 mucinous denocrcinoms, 2 denocrcinoms not otherwise specified, nd 1 neuroendocrine tumor. The uthors oserved men of 3.8 muttions per tumor (rnge 1 9). The most common ltertions were detected in KRAS (78%; 18 of 23 cses), TP53 (74%; 17 of 23), CDKN2A/B (35%; 8 of 23) nd SMAD4 (17%, 4 of 23). Muttions of PTEN were found in 13% of smples. The comprison of moleculr nlysis performed on FNA nd on mtched surgicl specimens showed perfect concordnce of the muttionl profile etween the two. In study of 29 EUS-FNA smples, Gleeson nd collegues 25 used 160-gene pnel to preopertively determine the muttionl lndscpe of 21 PDACs, 4 mpullry crcinoms, 1 Lynch syndrome ssocited PDAC (not previously treted with chemotherpy) nd 3 IPMNs, nd to verify its concordnce with tht otined from surgicl resection specimens. They demonstrted tht moderte to lrge trgeted NGS nlysis of preopertive EUS-FNA smples provides n excellent surrogte for the nlysis of surgiclly resected specimens to detect muttions ssocited with pncretic cncer. The min genes ltered were KRAS (93%; 27 of 29), TP53 (72%; 21 of 29), SMAD4 (31%; 9 of 29), nd GNAS (10%; 3 of 29). A TP53 muttion ws oserved frequently in ssocition with KRAS ltertion (95%; 20 of 21 TP53-mutted cses). Pthogenic SMAD4 ltertions were detected in out 30% of ptients with PDAC (7 of 21), ut in none of the ptients with mpullry crcinom. They reported n excellent concordnce etween muttions found in EUS- FNA specimens nd those detected in the pired surgicl mteril: in 15 of 18 cses the concordnce ws 100%. In 3 smples, they oserved some muttions in EUS-FNA specimens (in GRIN2A, GATA3, GNAS, nd KDM6A ut not in the surgicl specimens: ll discordnt muttions hd percentge of mutted lleles lower thn 14% nd were therefore consistent with suclonl moleculr events. Genetic Arch Pthol L Med Vol 142, April 2018 NGS of Pncretic EUS-FNA de Bise et l 459

3 Source, y Young et l, Tle 2. heterogeneity (including heterogeneity of KRAS muttions, n erly inititing event) hs een reported in pncretic cncers. 26,27 Considering tht preopertive smples contin only smll prt of the lesion, this represents limittion of ll preopertive dignostic procedures (including conventionl cytologic exmintion) in pncretic cncers s well s in cncers from other orgns. The proportion of mutted lleles identified y NGS represents n importnt clue to define muttion s suclonl. It cn e normlized to estimte the proportion of neoplstic cells crrying the muttion 28 nd should e tken into ccount for moleculr dignosis. In the work y Gleeson nd collegues, 25 s well s in our experience, moleculr heterogeneity represented relevnt issueinonlyminorityofpncretictumors. Siing Mulder nd collegues 29 lso reported good correltion etween muttions detected in preopertive FNA mteril nd those found in the mtched surgicl resection specimen. 29 Using pnel of 50 genes frequently Next-Genertion Sequencing Anlysis of Solid Pncretic Tumors Strting From Fine-Needle Aspirtion (FNA) Mteril Type of Tumor (No.) Genes Trgeted Pltform Mteril PDAC (18), P-NET (1), PC NOS (2) de Bise, PDAC (20), inoperle pncretic tumors (6), P-NET (5) Pnel of 287 cncerrelted genes nd 19 genes frequently rerrnged in cncer KRAS Di Mrco et l, PDAC (16) Whole-exome sequencing Kuot et l, SPN (7), PDAC (16), CTNNB1 P-NET (11) Dudley et l, Bile nd min pncretic duct smples: neoplstic (31), nonneoplstic (43) Custom pnel (39 Gleeson et l, PDAC (22), AA (4) Humn Comprehensive Cncer GeneRed DNAseq Trgeted Pnel V2 (pnel of 160 cncer Kmet et l, PDAC (27) AmpliSeq Cncer Pnel v2 (50 Siing Mulder et l, Vlero et l, PDAC (1) Unresectle PDAC (19) AmpliSeq Cncer Pnel V2 (50 Ion Ampliseq Comprehensive Cncer Pnel (409 More Frequent Altertions (%) HiSeq 2000 (Illumin) FNA KRAS (76.2), TP53 (71.4), CDKN2A/2B (38.1), SMAD4 (19.0), PTEN (14.3) 454 GS- Junior (Roche) HiScnSQ (Illumin) Fisher) c MiSeq (Illumin) HiSeq 2000 (Illumin) Fisher) Fisher) Fisher) EUS-guided FNA EUS-FNB PDAC nd inoperle pncretic tumors: KRAS (93.5) P-NET: KRAS (0) KRAS (93.7), TP53 (56.0), CDKN2A (50.0) EUS-FNA SPN: CTNNB1 (100) P-NET: CTNNB1 (9) PDAC: CTNNB1 (0) Brushing Neoplstic: KRAS (64.5), TP53 (45.2), SMAD4 (19.4), CDKN2A (12.9) Nonneoplstic: KRAS (2.3) EUS-FNA EUS-FNA EUS-FNA FNA KRAS (93.1), d TP53 (72.4), d SMAD4 (31.0), d GNAS (10.3) d KRAS (96.0), TP53 (44.0), CDKN2A (15.0), SMAD4 (11.0) KRAS, TP53, CDKN2A KRAS (73.7), TP53 (47.4), SMAD4 (31.6), ARID1 (15.8) Arevitions: AA, mpullry denocrcinom; EUS, endoscopic ultrsound guided; FNB, fine-needle iopsy; PC NOS, pncretic crcinoms not otherwise specified; PDAC, pncretic ductl denocrcinom; P-NET, pncretic neuroendocrine tumor; SPN, solid pseudoppillry neoplsm. Illumin Inc, Sn Diego, Cliforni. Roche Dignostics, Mnnheim, Germny. c Thermo Fisher Scientific, Wlthm, Msschusetts. d It is not possile to discriminte the muttion prevlence of solid versus cystic lesions: 3 intrductl ppillry mucinous neoplsms re included in the study (see Tle 3). mutted in cncer for the profiling of FNA mteril from the PDAC of 54-yer-old mn, they detected muttion in KRAS, TP53, SMAD4, nd CDKN2A strting from oth preopertive cytologic nd surgicl resection specimens. Next-genertion sequencing nlysis hs lso een successfully performed from pncretoiliry rush cytology smples, s demonstrted y Dudley nd collegues 30 in cohort of 81 ptients who underwent endoscopic retrogrde cholngiopncretogrphy. Also in this cohort of smples, KRAS muttions were the most frequent ltertion (26%; 21 of 81 cses nlyzed y NGS), nd TP53 ws the second most commonly mutted gene (17%; 14 of 81 cses). The uthors 30 oserved tht NGS is s sensitive s the nlysis of neuploidy y fluorescence in situ hyridiztion to preopertively identify pncretoiliry duct mlignncies. Intriguingly, in this pper KRAS muttions were detected lso in 2.3% (1 of 43) of nonneoplstic control smples. 30 It should e noted tht KRAS muttions hve een reported in 460 Arch Pthol L Med Vol 142, April 2018 NGS of Pncretic EUS-FNA de Bise et l

4 some cses of chronic pncretitis, where the presence of KRAS muttion hs een ssocited with evolution of the pncretitis to PDAC. 31,32 KRAS (74%; 14 of 19 cses), TP53 (47%; 9 of 19 cses), nd SMAD4 (32%; 6 of 19 cses) were lso the most frequently mutted genes in the study y Vlero nd collegues 33 performed on cohort of 19 FNAs from ptients with unresectle nonmetsttic pncretic tumors, using pnel of 409 genes. The uthors lso found muttions in ARID1 (16%; 3 of 19 cses), GRM8 (10%; 2 of 19 cses), nd TRIM33 (10%; 2 of 19 cses). They concluded tht somtic vrints identified in preopertive FNA smples using NGS my e used to guide the clinicl mngement of ptients with pncretic cncer. Differently from wht is oserved in PDAC, SPNs do not hve KRAS muttions, ut hror muttions in CTNNB1, the gene of the wnt pthwy encoding for -ctenin. Kuot et l 34 investigted CTNNB1 using NGS in cohort of SPNs, PDACs, nd pncretic neuroendocrine tumors (P-NETs) strting from EUS-FNA mteril. Muttions of CTNNB1 were detected in ll SPNs, 9% (1 of 11) of pncretic neuroendocrine tumors, nd no PDAC specimens. Even if the percentge of mutted lleles detected y NGS ws 20% or more ( percentge comptile with the nlyticl sensitivity of Snger sequencing), they were le to identify CTNNB1 muttion in only 1 of the smples using Snger sequencing. NGS OF EUS-FNA SAMPLES OF PANCREATIC CYST FLUID The studies tht hve ddressed NGS nlysis of EUS- FNA smples of cystic lesions re summrized in Tle 3. Pncretic cysts re heterogeneous group of lesions tht include injury nd inflmmtion relted conditions (~30% of cses) s well s neoplsms (~60% of cses). The lrge mjority of neoplstic cysts re of ductl linege, more frequently mucinous, IPMN, nd mucinous cystic neoplsm (MCN), ut lso of serous linege (serous cystdenom nd rrely cystdenocrcinom). 35 Al-Hddd nd collegues 36 hve shown tht the nlysis of KRAS nd loss of heterozygosity helps in the differentil dignosis of cystic mucinous pncretic lesions (IPMN nd MCN) when preopertive cytology is nondignostic or crcinoemryonic ntigen (CEA) cyst fluid levels re indeterminte. The iochemicl determintion of CEA is one of the most ccurte tumor mrkers to dignose mucinous pncretic cysts nd to distinguish them from nonmucinous cysts: in fct, high levels of CEA (.200 ng/ ml) strongly suggest mucinous neoplsi lthough reported cutoff CEA vlues vry considerly. 37,38 Severl studies hve now demonstrted tht highthroughput nlysis of multiple genetic mrkers with NGS pltforms dds useful informtion to tht otined fter the evlution of CEA nd cytologic specimens. Amto et l 39 nlyzed 51 cncer-ssocited genes using NGS in 48 IPMNs. The mrker more commonly ltered ws GNAS (79%; 38 of 48 cses). KRAS ws mutted in 50% of IPMNs (24 of 48), nd in 37.5% (18 of 48) the muttion coexisted with GNAS ltertions. Less frequently, muttions were found in TP53 (10%; 5 of 48 cses), BRAF (6%; 3 of 48 cses), nd CTNNB1 nd IDH1 (4%; 2 of 48 cses for ech gene). KRAS nd GNAS muttions coexisted in 37.5% of IPMNs (18 of 48). The mount of DNA otined from cyst fluid ws dequte for NGS (ie, t lest 20 ng of DNA) in 10 of 48 IPMNs. In these 10 smples, sequencing llowed detection of 10 of the 13 muttions found in the mtched surgicl specimens (6 of 7 GNAS muttions, 3 of 3 KRAS muttions, nd 1 of 2 TP53 muttions). 39 The comintion of GNAS nd KRAS preopertive testing ws lso demonstrted to e highly specific nd sensitive for IPMNs y Singhi et l. 40 Jones et l 41 nlyzed 92 cyst fluid smples using pnel of 39 genes. KRAS ws the gene most frequently mutted (47%; 43 of 92 cses), followed y GNAS (24%; 22 of 92 cses) nd CDKN2A (7%; 6 of 92 cses). Overll, 43% (40 of 92) of the smples did not show ny muttion in t lest 1 of the 39 genes in the pnel. In 71% (65 of 92) of the smples KRAS or GNAS muttion ws consistent with dignosis of IPMN y imging, in spite of low CEA levels. In one cse, n elevted level of CEA ws discordnt with the impression sed on imging, ut the finding of one KRAS muttion supported the preopertive dignosis of MCN. 41 The dt of the study supported the ssocition etween high-risk cysts nd ccumultion of genetic ltertions. KRAS nlysis provided useful informtion for the mlignncy risk in those cses tht would e dignosed s enign y imging nd in those with low CEA levels in the cyst fluid. The use of pnel of genes helped in detecting those muttions typiclly ssocited with mucinous lesions (KRAS, GNAS, CDKN2A) nd those dditionl chnges in genes (SMAD4, TP53) tht re ssocited with higher mlignncy risk nd/or with cysts feturing n infiltrting denocrcinom component. 41 Rosenum nd collegues (the sme group of Jones et l. 41 ) 42 studied 113 pncretic cystic lesions with pnel of 39 genes. The uthors found totl of 119 vrints in 67 smples. Most of them were muttions in KRAS (53%; 60 of 113 cses) or GNAS (24%; 27 of 113 cses). Other muttions were found in the following genes: CDKN2A (9%; 10 of 113 cses), TP53 (4%; 5 of 113 cses), nd SMAD4 (2%; 2 of 113 cses). Altertions in BRAF, NOTCH1, ndpik3ca were found in only 1 smple ech (0.9%; 1 of 113). 42 Considering only smples tht underwent surgicl resection (38; finl dignoses: 8 nonmucinous cysts, 6 IPMNs, nd 24 crcinoms), KRAS muttions were found in 75% (18 of 24) of the smples dignosed s cncer, 16% (1 of 6) of IPMNs, nd only in 12.5% (1 of 8) of nonmucinous cystic lesions. GNAS ltertions were found more frequently in IPMNs (33.3%; 3 of 6) thn in cncers (25.0%; 6 of 24) or nonmucinous cysts (0%; 0 of 8 cses). All the other ltertions (TP53, SMAD4, nd CDKN2A) were found only in cystic lesions with ssocited PDAC. Overll, the presence of KRAS muttions hd sensitivity nd specificity for cystic mucinous lesions (IPMNs or crcinoms) of 80% nd 88%, respectively. In contrst, GNAS muttions hd low sensitivity (27%) ut very high specificity (100%) for IPMN. In the study, the comintion of NGS with the nlysis of CEA in the cyst fluid reched 90% sensitivity nd 88% specificity for cystic mucinous lesions. 42 In retrospective lrge multicenter study, Springer nd collegues 43 showed tht the screening of cyst fluid for pnel of genetic mrkers (gene muttions, loss of heterozygosity, nd neuploidy), in conjunction with the clinicl fetures of the cyst, cn e used to firly ccurtely clssify cystic pncretic lesions nd to identify those cses tht require surgicl resection. The uthors nlyzed 11 genes (BRAF, CDKN2A, CTNNB1, GNAS, KRAS, NRAS, PIK3CA, RNF43, SMAD4, TP53, nd VHL) known to e mutted in cystic lesions in 130 cyst fluid smples. KRAS ws the most frequently mutted gene in IPMNs (78%; 75 of 96 cses) nd MCNs (50%; 6 of 12 cses), wheres it ws not ltered in serous cystdenoms nd SPNs. Serous cystdenoms Arch Pthol L Med Vol 142, April 2018 NGS of Pncretic EUS-FNA de Bise et l 461

5 Source, y Young et l, Tle 3. Next-Genertion Sequencing Anlysis of Cystic Pncretic Lesions Strting From Fine-Needle Aspirtion (FNA) Mteril Type of Tumor (No.) Genes Trgeted Pltform Mteril MCN (2, oth denocrcinoms) Pnel of 287 cncerrelted genes nd 19 genes frequently rerrnged in cncer Amto et l, IPMN (7) AmpliSeq Cncer Pnel v2 (50 More Frequent Altertions (%) HiSeq 2000 (Illumin) FNA KRAS (100.0), TP53 (100.0) Fisher) Cyst fluid GNAS, KRAS, TP53 c Wng et l, IPMN, MCN, PC NOS (17 totl) Noncoding RNA SoliD Fisher) Cyst fluid 15 mirnas differently expressed etween lowgrde nd highgrde lesions de Bise et l, Cyst NOS (26) KRAS 454 GS-Junior (Roche) d EUS-FNA KRAS: 38.5 (83.3 Springer et l, IPMN (96), MCN (12), SCA (12), SPN (10) Pnel of 11 custom genes Gleeson et l, IPMN (3) Humn Comprehensive Cncer GeneRed DNAseq Trgeted Pnel V2 (160 cncer genes pnel) Jones et l, Cyst NOS (92) Custom pnel (39 Rosenum et l, Cyst NOS (113) Custom pnel (39 in IPMN) MiSeq (Illumin) Cyst fluid IPMN: KRAS (78.0), GNAS (58.0), RNF43 (38.0) MCN: KRAS (50.0) SCA: VHL (42.0) SPN: CTNNB1 (100.0), PIK3CA (20.0) HiSeq 2000 (Illumin) EUS-FNA NA e Anchored multiplex PCR NGS pltform Anchored multiplex PCR NGS pltform EUS-FNA KRAS (47.0), GNAS (24.0) Cyst fluid Cncer: KRAS (75.0), GNAS (25.0), TP53 (16.7), CDKN2A (33.3) f IPMN: KRAS (100.0), GNAS (33.0) f Nonmucinous: KRAS (12.5) f Arevitions: Cyst NOS, cystic lesions not otherwise specified; EUS, endoscopic ultrsound guided; IPMN, intrductl ppillry mucinous neoplsm; MCN, mucinous cystic neoplsm; PC NOS, pncretic crcinoms not otherwise specified; SCA, serous cystdenom; SPN, solid pseudoppillry neoplsm. Illumin Inc, Sn Diego, Cliforni. Thermo Fisher Scientific, Wlthm, Msschusetts. c Dt out prevlence of muttions in the 7 IPMN cyst fluids re not ville. d Roche Dignostics, Mnnheim, Germny. e Dt out prevlence of muttions in the IPMN cyst fluids re not ville (see Tle 2). f Clculted on the 24 cysts with finl dignosis in the series; the studies y Jones et l 41 nd Rosenum et l 42 re from the sme group. frequently hd VHL muttions (42%; 5 of 12 cses), wheres ll 10 SPNs hrored muttions in CTNNB1. Overll, the uthors oserved tht when moleculr nd clinicl mrkers were comined, the sensitivity nd specificity in detecting serous cystdenom incresed to 100% nd 98%, respectively. For IPMN, the comintion of moleculr nd clinicl mrkers led to higher sensitivity when compred with composite moleculr mrkers lone (94% versus 76%), ut resulted in decrese in specificity (84% versus 97%). On the other hnd, the use of oth moleculr nd clinicl mrkers decresed sensitivity (90% versus 100%) ut incresed specificity (97% versus 75%) for the preopertive dignosis of MCN. For SPN, comining moleculr nd clinicl mrkers decresed the performnce of preopertive 462 Arch Pthol L Med Vol 142, April 2018 NGS of Pncretic EUS-FNA de Bise et l

6 Tle 4. Next-Genertion Sequencing (NGS) Anlysis of Pncretic Lesions Strting From Body Fluid Source, y Type of Tumor (No) Genes Trgeted Pltform Mteril More Frequent Altertions, % Zill et l, Advnced pncretoiliry Pnel of 54 custom HiSeq 2500 Blood KRAS (53.8), TP53 (38.5) crcinom (26) genes (Illumin) (cfdna) Yu et l, PDAC (34) Pnel of 9 custom genes NGS Fisher) c Pncretic juice KRAS (74.0), TP53 (59.0), GNAS (38.0), RNF43 (24.0), SMAD4 (15.0) Arevitions: cfdna, cell-free DNA; PDAC, pncretic ductl denocrcinom. Illumin Inc, Sn Diego, Cliforni. Results otined using oth NGS nd digitl polymerse chin rection. c Thermo Fisher Scientific, Wlthm, Msschusetts. ssessment: sensitivity decresed to 89% (from 100% using moleculr mrkers lone) nd specificity to 92% (100% using moleculr mrkers lone). 43 MicroRNAs re differentilly expressed in pncretic tumors. 44 Wng nd collegues 45 nlyzed y NGS pnel of micrornas in cohort of cyst fluid smples from ptients with mucinous cysts, IPMNs, nd pncretic cncers. The study showed tht multiple micrornas re differentilly undnt in high-grde invsive versus low-grde or enign lesions. Five micrornas (mir-125, mir-214, mir- 26, mir-30, nd mir-217) found to e differentilly expressed etween high-grde nd low-grde IPMNs y Wng et l 45 were lso detected s differentilly expressed y Mtthei et l 46 in IPMNs nlyzed using n rry-sed rel-time PCR method. in pncretic juice my distinguish ptients with PDCA from those with IPMNs nd disese controls. 47 Zill nd collegues 48 nlyzed with NGS cell-free DNA from the peripherl lood of 26 ptients with dvnced pncretic (n ¼ 18) or iliry (n ¼ 8) crcinoms. KRAS nd TP53 were the most frequently mutted genes, ut ltertions were commonly found lso in APC, SMAD4, FBXW7, nd BRAF genes. In the study, muttions of KRAS, TP53, APC, FBXW7, nd SMAD4 genes hd n verge sensitivity of 92.3%, specificity of 100%, nd dignostic ccurcy of 97.7% when compred with the muttion nlysis results from tumor iopsy smples, 48 thus demonstrting tht NGS nlysis of cell-free DNA llows detection of tumor-derived muttions in ptients with dvnced pncretoiliry crcinom. NEXT-GENERATION ANALYSIS OF OTHER FLUID BIOLOGICAL SPECIMENS FROM PATIENTS WITH PANCREATIC TUMORS Studies tht hve ddressed the NGS nlysis of fluid specimens other thn cyst fluid re summrized in Tle 4. Yu nd collegues 47 nlyzed the pncretic juice of ptients with PDAC (34 cses), ptients with IPMN (57 cses), nd controls with norml pncres or chronic pncretitis (totl of 24 cses). The nlysis ws performed using NGS for 9 genes (KRAS, GNAS, TP53, SMAD4, CDKN2A, RNF43, TGFBR2, BRAF, nd PIK3CA) nd digitl PCR to evlute the ccurcy of NGS. The pncretic juice of ptients with PDAC hd higher muttionl lod when compred with tht of ptients with IPMN (P ¼.003) nd tht of control groups (P,.001). 47 The most frequently mutted gene in PDAC pncretic juice ws KRAS (91%;31of34smples).Surprisingly,KRAS ws found to e mutted in 42% of controls (10 of 24 cses). 47 The second most common muttion in ptients with PDAC ws TP53 (58%; 20 of 34), which ws lso detected in ptients with IPMN (26%; 15 of 57), ut in none of the controls. In ddition to KRAS, GNAS nd RNF43 were lso sometimes mutted in control specimens: 17% (4 of 24 cses) nd 4% (1 of 24 cses), respectively. The uthors concluded tht the identifiction of mutted KRAS in pncretic juice is not specific enough to distinguish PDAC from IPMN or nonneoplstic controls. The most specific mrker ws SMAD4, mutted in only 1 of 80 cses without PDAC (n IPMN of 6 cm with intermeditegrde dysplsi), followed y TP53. The KRAS, SMAD4, nd TP53 muttions detected in smples collected efore the opertion were confirmed in the resection specimens. However, in few cses, muttions could not e demonstrted in the preopertive pncretic juice smples, nd were only identified in the surgicl resections. Thus, the preopertive moleculr nlysis of pncretic juice ppers to hve high positive ut low negtive predictive vlue. 47 According to the uthors, the concentrtion of TP53 nd SMAD4 muttions CONCLUSIONS AND FUTURE PERSPECTIVE In spite of the extensive knowledge out the moleculr ltertions of pncretic tumors ccumulted in the recent pst, the mortlity rte of PDAC ptients remins very high. As stted y the current Europen Society for Medicl Oncology guidelines, 49 to dte there re not trgetle molecules for personlized ptient tretment. Of the severl mrkers investigted, KRAS remins the one most commonly used for single-gene testing, lthough its use is gretly limited y the identifiction of mutted KRAS in out 10% of chronic pncretitis nd/or low-grde pncretoiliry epithelil cell dysplsi (in some studies frequencies.10% hve een reported) Thus, the Ppnicolou Society of Cytopthology guidelines do not support KRAS testing of solid pncretic msses nd ile duct strictures s useful single-gene ncillry test. The sme guidelines report tht numer of gene muttions (KRAS, GNAS, VHL, RNF43 nd CTNNB1) my e of id in the identifiction of specific cystic neoplsms. 50 Given this context, NGS my e instrumentl for the preopertive moleculr dignosis of pncretic lesions, ecuse it offers the opportunity of screening simultneously wide numer of muttions while using smll mounts of DNA. It is ecoming cler tht the use of wide gene pnels increses clinicl sensitivity nd specificity, minimizing the risk of flse-positive results. High nlyticl sensitivity nd positive predictive vlue cn prevent repet iopsies, nd thus improve preopertive dignosis nd preopertive ptient risk strtifiction nd mngement, while reducing costs. On the other hnd, NGS requires expensive instrumenttion nd specilized expertise, nd its ppliction to the preopertive dignosis needs roust vlidtion in lrge multicenter series of pired preopertive nd surgicl smples. Further studies, with creful evlution of costs versus enefits, will e necessry efore the full implementtion of NGS in clinicl prctice. Arch Pthol L Med Vol 142, April 2018 NGS of Pncretic EUS-FNA de Bise et l 463

7 This work ws supported in prt y Itlin Government- Ministero Dell Slute grnt RF to G.T. References 1. Ryn DP, Hong TS, Brdeesy N. Pncretic denocrcinom. N Engl J Med. 2014;371(11): Hong SK, Loren DE, Rogrt JN, et l. Trgeted cyst wll puncture nd spirtion during EUS-FNA increses the dignostic yield of premlignnt nd mlignnt pncretic cysts. Gstrointest Endosc. 2012;75(4): Dumonceu JM, Polkowski M, Lrghi A, et l. Indictions, results, nd clinicl impct of endoscopic ultrsound (EUS)-guided smpling in gstroenterology: Europen Society of Gstrointestinl Endoscopy (ESGE) clinicl guideline. Endoscopy. 2011;43(10): Jenssen C, Hocke M, Fusroli P, et l. EFSUMB Guidelines on Interventionl Ultrsound (INVUS), prt IV EUS-guided interventions: generl spects nd EUS-guided smpling (long version). Ultrschll Med. 2016;37(2): E33 E Hwss NE. Compring the sensitivities nd specificities of two dignostic procedures performed on the sme group of ptients. Br J Rdiol. 1997;70(832): Vrdrjulu S, Fockens P, Hwes RH. Best prctices in endoscopic ultrsound-guided fine-needle spirtion. Clin Gstroenterol Heptol. 2012; 10(7): Eser S, Schnieke A, Schneider G, Sur D. Oncogenic KRAS signlling in pncretic cncer. Br J Cncer. 2014;111(5): Icouzio-Donhue CA. Genetic evolution of pncretic cncer: lessons lernt from the pncretic cncer genome sequencing project. Gut. 2012;61(7): Khlid A, Nodit L, Zhid M, et l. Endoscopic ultrsound fine needle spirte DNA nlysis to differentite mlignnt nd enign pncretic msses. Am J Gstroenterol. 2006;101(11): Td M, Komtsu Y, Kwe T, et l. Quntittive nlysis of K-rs gene muttion in pncretic tissue otined y endoscopic ultrsonogrphy-guided fine needle spirtion: clinicl utility for dignosis of pncretic tumor. Am J Gstroenterol. 2002;97(9): Khlid A, Dewitt J, Ohori NP, et l. EUS-FNA muttionl nlysis in differentiting utoimmune pncretitis nd pncretic cncer. Pncretology. 2011;11(5): Goggins M. Identifying moleculr mrkers for the erly detection of pncretic neoplsi. Semin Oncol. 2007;34(4): de Bise D, Visni M, Bccrini P, et l. Next genertion sequencing improves the ccurcy of KRAS muttion nlysis in endoscopic ultrsound fine needle spirtion pncretic lesions. PLoS One. 2014;9(2):e Dillon DA, Johnson CC, Topzin MD, Tllini G, Rimm DL, Cost JC. The utility of Ki-rs muttion nlysis in the cytologic dignosis of pncretoiliry neoplsm. Cncer J. 2000;6(5): Jones S, Zhng X, Prsons DW, et l. Core signling pthwys in humn pncretic cncers reveled y glol genomic nlyses. Science. 2008; 321(5897): Di Mrco M, Astolfi A, Grssi E, et l. Chrcteriztion of pncretic ductl denocrcinom using whole trnscriptome sequencing nd copy numer nlysis y single-nucleotide polymorphism rry. Mol Med Rep. 2015;12(5): Ling WS, Crig DW, Crpten J, et l. Genome-wide chrcteriztion of pncretic denocrcinom ptients using next genertion sequencing. PLoS One. 2012;7(10):e Fores SA, Bere D, Boutselkis H, et l. COSMIC: somtic cncer genetics t high-resolution. Nucleic Acids Res. 2017;45(D1):D777 D L Ros S, Sess F, Cpell C. Acinr cell crcinom of the pncres: overview of clinicopthologic fetures nd insights into the moleculr pthology. Front Med (Lusnne). 2015;2: Chen M, Vn Ness M, Guo Y, Gregg J. Moleculr pthology of pncretic neuroendocrine tumors. J Gstrointest Oncol. 2012;3(3): Reid MD, Choi H, Blci S, Akks G, Adsy V. Serous cystic neoplsms of the pncres: clinicopthologic nd moleculr chrcteristics. Semin Dign Pthol. 2014;31(6): de Bise D, de Luc C, Grgnno G, et l. Fully utomted PCR detection of KRAS muttions on pncretic endoscopic ultrsound fine-needle spirtes [pulished online hed of print April 27, 2016]. J Clin Pthol. doi: / jclinpth Kmet E, Sugimori K, Kneko T, et l. Dignosis of pncretic lesions collected y endoscopic ultrsound-guided fine-needle spirtion using nextgenertion sequencing. Oncol Lett. 2016;12(5): Young G, Wng K, He J, et l. Clinicl next-genertion sequencing successfully pplied to fine-needle spirtions of pulmonry nd pncretic neoplsms. Cncer Cytopthol. 2013;121(12): Gleeson FC, Kerr SE, Kipp BR, et l. Trgeted next genertion sequencing of endoscopic ultrsound cquired cytology from mpullry nd pncretic denocrcinom hs the potentil to id ptient strtifiction for optiml therpy selection. Oncotrget. 2016;7(34): Lghi L, Oretegli O, Binchi P, et l. Common occurrence of multiple K- RAS muttions in pncretic cncers with ssocited precursor lesions nd in iliry cncers. Oncogene. 2002;21(27): Visni M, de Bise D, Bccrini P, et l. Multiple KRAS muttions in pncretic denocrcinom: moleculr fetures of neoplstic clones indicte the selection of divergent popultions of tumor cells. Int J Surg Pthol. 2013;21(6): de Bise D, Cesri V, Visni M, et l. High-sensitivity BRAF muttion nlysis: BRAF V600E is cquired erly during tumor development ut is heterogeneously distriuted in suset of ppillry thyroid crcinoms. J Clin Endocrinol Met. 2014;99(8):E1530 E Siing Mulder BG, Mieog JS, Hndgrf HJ, et l. Trgeted nextgenertion sequencing of FNA-derived DNA in pncretic cncer. J Clin Pthol. 2017;70(2): Dudley JC, Zheng Z, McDonld T, et l. Next-genertion sequencing nd fluorescence in situ hyridiztion hve comprle performnce chrcteristics in the nlysis of pncreticoiliry rushings for mlignncy. J Mol Dign. 2016; 18(1): Arvnitkis M, Vn Lethem JL, Prm J, De Merteler V, Delhye M, Deviere J. Predictive fctors for pncretic cncer in ptients with chronic pncretitis in ssocition with K-rs gene muttion. Endoscopy. 2004;36(6): Kmisw T, Tkum K, Tt T, Egw N, Ymguchi T. Long-term follow-up of chronic pncretitis ptients with K-rs muttion in the pncretic juice. Heptogstroenterology. 2011;58(105): Vlero V 3rd, Sunders TJ, He J, et l. Relile detection of somtic muttions in fine needle spirtes of pncretic cncer with next-genertion sequencing: implictions for surgicl mngement. Ann Surg. 2016;263(1): Kuot Y, Kwkmi H, Ntsuizk M, et l. CTNNB1 muttionl nlysis of solid-pseudoppillry neoplsms of the pncres using endoscopic ultrsoundguided fine-needle spirtion nd next-genertion deep sequencing. J Gstroenterol. 2015;50(2): Bsturk O, Con I, Adsy NV. Pncretic cysts: pthologic clssifiction, differentil dignosis, nd clinicl implictions. Arch Pthol L Med. 2009; 133(3): Al-Hddd M, DeWitt J, Shermn S, et l. Performnce chrcteristics of moleculr (DNA) nlysis for the dignosis of mucinous pncretic cysts. Gstrointest Endosc. 2014;79(1): vn der Wij LA, vn Dullemen HM, Porte RJ. Cyst fluid nlysis in the differentil dignosis of pncretic cystic lesions: pooled nlysis. Gstrointest Endosc. 2005;62(3): Oh HC, Kng H, Brugge WR. Cyst fluid mylse nd CEA levels in the differentil dignosis of pncretic cysts: single-center experience with histologiclly proven cysts. Dig Dis Sci. 2014;59(12): Amto E, Molin MD, Mfficini A, et l. Trgeted next-genertion sequencing of cncer genes dissects the moleculr profiles of intrductl ppillry neoplsms of the pncres. J Pthol. 2014;233(3): Singhi AD, Nikiforov MN, Fsnell KE, et l. Preopertive GNAS nd KRAS testing in the dignosis of pncretic mucinous cysts. Clin Cncer Res. 2014;20(16): Jones M, Zheng Z, Wng J, et l. Impct of next-genertion sequencing on the clinicl dignosis of pncretic cysts. Gstrointest Endosc. 2016;83(1): Rosenum MW, Jones M, Dudley JC, Le LP, Ifrte AJ, Pitmn MB. Nextgenertion sequencing dds vlue to the preopertive dignosis of pncretic cysts. Cncer. 2017;125(1): Springer S, Wng Y, Dl Molin M, et l. A comintion of moleculr mrkers nd clinicl fetures improve the clssifiction of pncretic cysts. Gstroenterology. 2015;149(6): Müller S, Rulefs S, Bruns P, et l. Next-genertion sequencing revels novel differentilly regulted mrnas, lncrnas, mirnas, sdrnas nd pirna in pncretic cncer. Mol Cncer. 2015;14: Wng J, Pris PL, Chen J, et l. Next genertion sequencing of pncretic cyst fluid micrornas from low grde-enign nd high grde-invsive lesions. Cncer Lett. 2015;356(2, pt B): Mtthei H, Wylie D, Lloyd MB, et l. mirna iomrkers in cyst fluid ugment the dignosis nd mngement of pncretic cysts. Clin Cncer Res. 2012;18(17): Yu J, Sdkri Y, Shindo K, et l. Digitl next-genertion sequencing identifies low-undnce muttions in pncretic juice smples collected from the duodenum of ptients with pncretic cncer nd intrductl ppillry mucinous neoplsms. Gut. 2017;66(9): Zill OA, Greene C, Seisnovic D, et l. Cell-free DNA next-genertion sequencing in pncretoiliry crcinoms. Cncer Discov. 2015;5(10): Ducreux M, Cuhn AS, Crmell C, et l. Cncer of the pncres: ESMO clinicl prctice guidelines for dignosis, tretment nd follow-up. Ann Oncol 2015;26(suppl 5):v56 v Lyfield LJ, Ehy H, Filie AC, et l. Utiliztion of ncillry studies in the cytologic dignosis of iliry nd pncretic lesions: the Ppnicolou Society of Cytopthology guidelines for pncretoiliry cytology. Dign Cytopthol. 2014; 42(4): Luttges J, Reinecke-Luthge A, Mollmnn B, et l. Duct chnges nd K-rs muttions in the disese-free pncres: nlysis of type, ge reltion nd sptil distriution. Virchows Arch. 1999;435(5): Lohr M, Kloppel G, Misonneuve P, Lowenfels AB, Luttges J. Frequency of K-rs muttions in pncretic intrductl neoplsis ssocited with pncretic ductl denocrcinom nd chronic pncretitis: met-nlysis. Neoplsi. 2005;7(1): Lohr M, Misonneuve P, Lowenfels AB. K-rs muttions nd enign pncretic disese. Int J Pncretol. 2000;27(2): Arch Pthol L Med Vol 142, April 2018 NGS of Pncretic EUS-FNA de Bise et l