RNA Sequencing Identifies Frequent Mitogen-activated Protein Kinase–associated Fusion Genes in Intraductal Tubulopapillary Neoplasms of the Pancreas (2023)

Intraductal tubulopapillary neoplasms (ITPNs) of the pancreas are rare cystic precursor neoplasms to invasive pancreatic ductal adenocarcinoma (PDAC) and account for 3% of intraductal neoplasms of the pancreas. Originally reported by Yamaguchi et al,1Yamaguchi H. et al.Am J Surg Pathol. 2009; 33: 1164-1172Crossref PubMed Scopus (149) Google Scholar the clinicopathologic features of ITPNs are distinct from other pancreatic cysts. Radiographically, ITPNs are characterized by a nodular mass within dilated pancreatic ducts (Supplementary Figure 1A and B). These nodules are histologically composed of a tubulopapillary growth of epithelium with scant cytoplasmic mucin and necrotic foci (Supplementary Figure 1C–E).2Basturk O. et al.Am J Surg Pathol. 2017; 41: 313-325Crossref PubMed Scopus (63) Google Scholar In addition to their architectural complexity, ITPNs uniformly exhibit high-grade cytologic atypia and, in most cases, have an associated invasive carcinoma component (Supplementary Figure 1F–H). Although the morphologic features of ITPNs are clearly defined, the pathogenesis of these neoplasms remains poorly understood. The genomic findings of ITPNs differ significantly from those that are typically seen in other cystic precursor neoplasms of the pancreas and PDAC. Mutations in KRAS and GNAS are often absent in ITPNs, whereas these genomic alterations are frequently detected in intraductal papillary mucinous neoplasms and associated PDAC. Other than a subgroup of ITPNs harboring FGFR2 fusion genes and mutations within chromatin remodeling genes (eg, BAP1) and genes associated with the mechanistic target of rapamycin signaling pathway (eg, PIK3CA), recurrent driver genes have yet to be described in ITPNs.3Yamaguchi H. et al.Am J Surg Pathol. 2011; 35: 1812-1817Crossref PubMed Scopus (79) Google Scholar,4Basturk O. et al.Mod Pathol. 2017; 30: 1760-1772Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar In fact, some ITPNs do not harbor alterations in genes associated with PDAC. Considering KRAS wild-type PDACs often harbor alternative drivers of the mitogen-activated protein kinase (MAPK) signaling pathway, we performed both DNA- and RNA-based targeted next-generation sequencing (NGS) and whole transcriptome sequencing (WTS) of 23 surgically resected ITPNs (Supplementary Material and Methods). Consistent with previous studies, targeted NGS detected KRAS missense mutations in a minority of ITPNs (n = 2, 9%) (Table 1). However, alterations in genes coding for members of the MAPK signaling pathway were also identified and included a BRAF missense mutation (n = 1), ERBB2 amplification (n = 1), STRN-ALK fusion gene (n = 1), BRAF fusion genes (SND1-BRAF and AGK-BRAF, n = 2), ATP1B1-NRG1 fusion gene (n = 2), TRIM24-RET (n = 1), and multiple FGFR2 fusion genes (FGFR2-BICC1, FGFR2-CIT, FGFR2-FAM76A, FGFR2-PPHLN1, and FGFR2-NOL4; n = 5). Fifteen of 23 ITPNs (65%) had MAPK driver alterations and 11 ITPNs (48%) harbored fusion genes, which were mutually exclusive from each other. Additional alterations in decreasing order occurred in TP53 (n = 10), SMAD4 (n = 9), CDKN2A (n = 7), chromatin remodeling genes (BAP1 and ARID1A, n = 5), homologous recombination-related genes (BRCA2 and CHEK2, n = 3), STK11 (n = 2), Myc family of oncogenes (MYC and NMYC, n = 2), and PTEN (n = 1). Moreover, multiple chromosomal abnormalities were identified in 10 ITPNs (43%).Table 1Clinicopathologic Findings of 23 Patients With an ITPN of the PancreasCase no.Age (y)SexLocationSize (cm)Associated carcinomaGene fusionOther genomic alterations164MPancreatic head1.1YesNoneKRAS p.G12V, TP53 p.R248W, TP53 deletion, CDKN2A/B deletion, STK11 deletion, MYCN amplification (25 copies)275MPancreatic body and tail7.0YesNoneKRAS p.G12R, TP53 p.L194R, TP53 deletion, CDKN2A/B deletion, SMAD4 deletion, chromosomal gain of 11q, chromosomal loss of 16p364FPancreatic body2.7YesNoneBRAF p.V600E, chromosomal gain of 1q, chromosomal loss of 1p469FPancreatic body and tail9.0YesNoneERBB2 amplification (19 copies), TP53 p.T125_splice, ARID1A p.A245Gfs∗113534MPancreatic head7.5YesSTRN-ALKTP53 deletion, SMAD4 deletion6aIn addition to the patient's surgical specimen, the corresponding preoperative specimen (pancreatic cyst fluid) was molecularly evaluated.62MPancreatic head3.8NoSND1-BRAFbThe gene fusion was also detected within the patient's corresponding preoperative pancreatic cyst fluid.TP53 deletion718FEntire pancreas18.0NoAGK-BRAFTP53 deletion, SMAD4 deletion, chromosomal gain of 7p and 7q, chromosomal loss of 6p and 6q8aIn addition to the patient's surgical specimen, the corresponding preoperative specimen (pancreatic cyst fluid) was molecularly evaluated.59MPancreatic body and tail3.5YescThe gene fusion was also detected within the ITPN-associated invasive PDAC.ATP1B1-NRG1bThe gene fusion was also detected within the patient's corresponding preoperative pancreatic cyst fluid.SMAD4 deletion979MEntire pancreas26.6YesATP1B1-NRG1SMAD4 deletion, CHEK2 p.S372F, chromosomal loss of 1p, 6q, and 16p10aIn addition to the patient's surgical specimen, the corresponding preoperative specimen (pancreatic cyst fluid) was molecularly evaluated.68FPancreatic body and tail6.4YescThe gene fusion was also detected within the ITPN-associated invasive PDAC.TRIM24-RETbThe gene fusion was also detected within the patient's corresponding preoperative pancreatic cyst fluid.CDKN2A/B deletion, SMAD4 deletion, chromosomal gain of 1q and 12q, chromosomal loss of 1p and 14q1161FPancreatic head5.0YescThe gene fusion was also detected within the ITPN-associated invasive PDAC.FGFR2-BICC1CDKN2A/B deletion1227FPancreatic head3.5YesFGFR2-CITBAP1 p.F46Lfs∗26, CDKN2A/B deletion13aIn addition to the patient's surgical specimen, the corresponding preoperative specimen (pancreatic cyst fluid) was molecularly evaluated.81FPancreatic body3.9NoFGFR2-TACC2bThe gene fusion was also detected within the patient's corresponding preoperative pancreatic cyst fluid.TP53 p.R273H, MYC amplification (16 copies)1473MPancreatic head8.0YesFGFR2-FAM76ATSC1 p.E1101∗1563MPancreatic body and tail7.5YesFGFR2-PPHLN1BAP1 splice site c.376-33_378del36, MYC amplification (8 copies)1678FPancreatic tail5.4YescThe gene fusion was also detected within the ITPN-associated invasive PDAC.FGFR2-TACC2SMAD4 deletion, ARID1A p.Q581∗, ARID1A deletion, chromosomal loss of 9p, 9q, 12q, and 14q1722MEntire pancreas13.4YesFGFR2-NOL4BAP1 splice site c.428-2A>G, CDKN2A/B deletion, BRCA2 deletion1853FEntire pancreas12.5NoCFTR-RASGRF2MYC amplification (7 copies), chromosomal gain of 1p1937FPancreatic head4.8YesSPTLC1-JAK2SMAD4 deletion, STK11 p.G196Afs∗912074MEntire pancreas10.5YesATXN3-MAML3TP53 p.E346∗, TP53 deletion, CDKN2A/B deletion, RB1 p.E580∗, RB1 deletion, PTEN p.D107Y, PTEN deletion2168FPancreatic head6.0YesNCOA3-MAML2TP53 p.S241F, TP53 deletion, CDKN2A/B deletion, SMAD4 deletion, chromosomal loss of 11p, 11q, 13q, 19p, and 19q2273FPancreatic head3.1YesRUNX2-MAML2TP53 deletion, CDKN2A/B deletion, BRCA2 p.K2674∗, BRCA2 deletion, chromosomal loss of 17q2347MPancreatic head5.0YesATXN3-MAML2ARID1A deletion, chromosomal loss of 11p and 11qa In addition to the patient's surgical specimen, the corresponding preoperative specimen (pancreatic cyst fluid) was molecularly evaluated.b The gene fusion was also detected within the patient's corresponding preoperative pancreatic cyst fluid.c The gene fusion was also detected within the ITPN-associated invasive PDAC. Open table in a new tab Considering the relative abundance of MAPK-associated fusion genes that were found by targeted NGS, we hypothesized that additional fusion genes may be present among the 8 ITPNs (35%) without a discernable MAPK driver alteration. Therefore, we performed WTS and detected fusion genes in the remaining 8 ITPNs: FGFR2-TACC2 (n = 2), CFTR-RASGRF2 (n = 1), SPTLC1-JAK2 (n = 1), ATXN3-MAML3 (n = 1), NCOA3-MAML2 (n = 1), RUNX2-MAML2 (n = 1), and ATXN3-MAML2 (n = 1) (Supplementary Figure 2). Analogous to the results of targeted NGS, each fusion gene was mutually exclusive from other fusion genes, thus suggesting their involvement in common pathologic mechanisms. Because many of these fusion genes have not been described in other neoplasms, fluorescence in situ hybridization using MAML2 break-apart probes (orange, 5′ end of MAML2; green, 3′ end of MAML2) was performed on cases 21, 22, and 23 and confirmed the presence of a MAML2 rearrangement (Supplementary Figure 3). Additionally, the ITPN-associated PDAC for cases 8, 10, 11, and 16 were evaluated by either targeted NGS or WTS, and the respective fusion identified within the ITPN was also detected. Matched preoperative pancreatic cyst fluid specimens were available for a subset of ITPNs (cases 6, 8, 10, and 13), and once again the respective fusion genes were identified. Overall, 17 of 23 ITPNs (74%) were found to have MAPK driver alterations that included fusion genes and were mutually exclusive from each other. These fusion genes were also present within corresponding ITPN-associated PDACs and detected in preoperative pancreatic cyst fluid. Among the remaining 6 ITPNs, novel fusion genes were identified that were once again mutually exclusive from other fusion genes and included the following: CFTR-RASGRF2, SPTLC1-JAK2, ATXN3-MAML3, NCOA2-MAML2, RUNX2-MAML2, and ATXN3-MAML2. Fusion genes involving RASGRF2 and JAK2, but with different 5′ partners, have been previously reported in melanocytic lesions and hematologic neoplasms, respectively, and shown to result in downstream MAPK activation.5Houlier A. et al.Pigment Cell Melanoma Res. 2021; 34: 1074-1083Crossref PubMed Scopus (11) Google Scholar,6Peeters P. et al.Blood. 1997; 90: 2535-2540Crossref PubMed Google Scholar Thus, although the CFTR-RASGRF2 and SPTLC1-JAK2 fusion genes have not been specifically described, these alterations likely represent genomic drivers of the MAPK signaling pathway. In further support that these fusion genes similarly affect the MAPK signaling pathway, WTS of 9 ITPNs (Supplementary Figure 4) identified analogous gene expression profiles among each ITPN; however, the profiles were distinctly different compared with 13 intraductal papillary mucinous neoplasms and further underscore that ITPNs are a separate neoplasm from intraductal papillary mucinous neoplasms. The presence of MAML2 and MAML3 fusion genes in ITPNs is an intriguing finding. Both MAML2 and MAML3 belong to the 3-member transcriptional coactivator MAML (mastermind-like) family that plays an essential role in the transcriptional activation of multiple signaling pathways. However, our current understanding of the MAML proteins in cancer, such as mucoepidermoid carcinomas (MECs) of the salivary gland, is largely based on studies of the CRTC1-MAML2 fusion gene. The generation of a genetically engineered mouse model has established CRTC1-MAML2 as a key oncoprotein in the pathogenesis of MECs.7Chen Z. et al.JCI Insight. 2021; 6: e139497Crossref PubMed Scopus (31) Google Scholar Further, both in vitro cell line experiments and protein expression analysis of primary MECs have demonstrated that the CRTC1-MAML2 fusion gene is associated with MAPK activation.8Chen Z. et al.Oncogene. 2014; 33: 3869-3877Crossref PubMed Scopus (73) Google Scholar Blockade of the MAPK pathway with anti–epidermal growth factor receptor antibodies or pharmacologic inhibitors decreases cell growth and survival in CRTC1-MAML2 fusion-positive MEC cell lines and human MEC xenografts. It is therefore reasonable to speculate that the ITPN-associated MAML2 and MAML3 fusion genes are oncogenic MAPK driver alterations. There are limitations to our study. Although it represents one of the largest series of pancreatic ITPNs to be molecularly analyzed, the number of cases evaluated is relatively small, and concluding that driver alterations, especially fusion genes, within the MAPK signaling pathway are a universal feature of ITPNs may be premature. It is important to note, however, that ITPNs can arise from the bile duct and also represent precursor neoplasms to cholangiocarcinoma.9Park H.J. et al.Pathol Int. 2010; 60: 630-635Crossref PubMed Scopus (27) Google Scholar Sequencing studies of biliary ITPNs are limited, but mutations in KRAS and BRAF are of similar low prevalence in biliary ITPNs as pancreatic ITPNs.10Gross C. et al.Cancers. 2021; 13: 2742Crossref PubMed Scopus (9) Google Scholar Moreover, FGFR2 alterations have been found in ITPNs of the bile duct. Considering the radiographic, histologic, and molecular parallels between pancreatic and biliary ITPNs, it is plausible that biliary ITPNs are also characterized by frequent MAPK-associated fusion genes. Unfortunately, WTS studies of biliary ITPNs have not been reported. Another issue with our study is the lack of orthogonal confirmation of all fusion genes described herein. Many fusion genes detected by targeted NGS were validated through WTS (Supplementary Material and Methods), and break-apart fluorescence in situ hybridization was used to confirm the presence of MAML2 rearrangements. Additionally, the identification of the same fusion gene in matched preoperative pancreatic cyst fluid and corresponding ITPN-associated PDACs provides further support to the validity of our findings. In summary, we report the results of targeted NGS and WTS of pancreatic ITPNs, and, consistent with previous findings, missense mutations in KRAS were seen in some cases. Notably, other genomic alterations implicated within the MAPK signaling pathway were identified in the remaining cases and were mutually exclusive from each other. The predominant genomic alteration found in ITPNs was the presence of a fusion gene, many of which have not been described in PDAC or other neoplasms. Finally, the detection of ITPN-associated fusion genes in preoperative pancreatic cyst fluid suggests these genomic alterations could potentially improve the early detection of these cystic precursor neoplasms to PDAC. Members of the Pancreatic Cyst Alliance are as follows: James Tucker, PhD,1 Amer H. Zureikat, MD,2 Jin He, MD, PhD,3 Alessandro Paniccia, MD,2 Kenneth K. Lee, MD,2 Herbert J. Zeh, MD,4 Melissa E. Hogg, MD,5 Anil K. Dasyam, MD,6 Kevin McGrath, MD,7 Anne Marie Lennon, MD, PhD,8 Kenneth E. Fasanella, MD,7 Elham Afghani, MD, MPH,8 Randall E. Brand, MD,7 Adam Slivka, MD, PhD,7 Nisa Kubiliun, MD,9 Christopher J. VandenBussche, MD, PhD,10 Elizabeth D. Thompson, MD, PhD,10 Michael S. Torbenson, MD,11 Daniela S. Allende, MD,12 Phoenix D. Bell, MD,4 Cihan Kaya, PhD,1 and Abigail I. Wald, PhD1; from the 1Department of Pathology, University of Pittsburgh Medical Center, Pittsburgh, Pennsylvania; 2Department of Surgery, University of Pittsburgh Medical Center, Pittsburgh, Pennsylvania; 3Department of Surgery, Johns Hopkins University School of Medicine, Baltimore, Maryland; 4Department of Surgery, University of Texas Southwestern, Dallas, Texas; 5Department of Surgery, NorthShore University Health System, Evanston, Illinois; 6Department of Radiology, University of Pittsburgh Medical Center, Pittsburgh, Pennsylvania; 7Department of Medicine, University of Pittsburgh Medical Center, Pittsburgh, Pennsylvania; 8Department of Medicine, Johns Hopkins University School of Medicine, Baltimore, Maryland; 9Department of Medicine, University of Texas Southwestern, Dallas, Texas; 10Department of Pathology, Sol Goldman Pancreatic Cancer Research Center, Johns Hopkins University School of Medicine, Baltimore, Maryland; 11Department of Laboratory Medicine and Pathology, Mayo Clinic Rochester, Rochester, Minnesota; and 12Department of Anatomic Pathology, Cleveland Clinic, Cleveland, Ohio. Ralph H. Hruban, MD (Conceptualization: Equal; Data curation: Equal; Formal analysis: Equal; Writing – original draft: Equal; Writing – review & editing: Equal). Lodewijk A. A. Brosens, MD, PhD (Data curation: Supporting). Vincenzo Condello, PhD (Data curation: Supporting). James Tucker, PhD (Data curation: Supporting). Amer H. Zureikat, MD (Data curation: Supporting). Jin He, MD, PhD (Data curation: Supporting). Alessandro Paniccia, MD (Data curation: Supporting). Kenneth K. Lee, MD (Data curation: Supporting). Herbert J. Zeh, MD (Data curation: Supporting). Melissa E. Hogg, MD (Data curation: Supporting). Anil K. Dasyam, MD (Data curation: Supporting). Kevin McGrath, MD (Data curation: Supporting). Anne Marie Lennon, MD, PhD (Data curation: Supporting). Kenneth E. Fasanella, MD (Data curation: Supporting). Elham Afghani, MD, MPH (Data curation: Supporting). Randall E. Brand, MD (Data curation: Supporting). Adam Slivka, MD, PhD (Data curation: Supporting). Nisa Kubiliun, MD (Data curation: Supporting). Christopher J. VandenBussche, MD, PhD (Data curation: Supporting). Elizabeth D. Thompson, MD, PhD (Data curation: Supporting). Michael S. Torbenson, MD (Data curation: Supporting). Daniela S. Allende, MD (Data curation: Supporting). Phoenix D. Bell, MD (Data curation: Supporting). Cihan Kaya, PhD (Data curation: Supporting). Abigail I. Wald, PhD (Conceptualization: Equal; Data curation: Equal; Formal analysis: Equal; Writing – original draft: Equal; Writing – review & editing: Equal). Marina N. Nikiforova, MD (Conceptualization: Equal; Data curation: Equal; Formal analysis: Equal; Writing – original draft: Equal; Writing – review & editing: Equal). Aatur Singhi, MD, PhD (Conceptualization: Lead; Data curation: Lead; Formal analysis: Lead; Writing – original draft: Lead; Writing – review & editing: Lead). Study approval was obtained from the authors’ respective institutional review boards. The anatomic pathology surgical archives from the departments of pathology at the University of Pittsburgh Medical Center (UPMC), Johns Hopkins Hospital, and University Medical Center Utrecht were queried for the diagnosis of ITPN. Cases with hematoxylin and eosin–stained slides and corresponding formalin-fixed paraffin-embedded tissue were retrieved and reviewed to confirm the diagnosis of an ITPN by a trained surgical pathologist with gastrointestinal tract and hepatopancreatobiliary subspecialty training (J.W.L., R.H.H., L.A.A.B., and/or A.D.S.). The diagnosis of an ITPN was based on the 2010 and 2019 World Health Organization classification of tumors of the digestive system.1Adsay N.V. et al.Intraductal neoplasm of the pancreas.in: WHO classification of tumors of the digestive system. 4th ed. IARC Press, Lyon2010: 304-313Google Scholar, 2Basturk O. et al.Pancreatic intraductal tubulopapillary neoplasm.in: WHO classification of tumors of the digestive system. 5th ed. IARC Press, Lyon2019: 317-318Google Scholar, 3Yamaguchi H. et al.Am J Surg Pathol. 2009; 33: 1164-1172Crossref PubMed Scopus (160) Google Scholar Twenty-three ITPNs were identified as well as 4 matched preoperative pancreatic cyst fluid specimens that were submitted for molecular testing (PancreaSeqV2) through the Molecular and Genomic Pathology Laboratory at UPMC.4Singhi A.D. et al.Gut. 2018; 67: 2131-2141Crossref PubMed Scopus (254) Google Scholar Nucleic acid extraction and subsequent DNA- and RNA-based targeted NGS were performed within the Clinical Laboratory Improvement Amendments–certified and College of American Pathologists–accredited Molecular and Genomic Pathology Laboratory at UPMC. Genomic DNA and RNA were from formalin-fixed paraffin-embedded tissue and pancreatic cyst fluid was obtained by using the and on or the Nucleic on DNA and RNA were on the using the and the RNA testing from DNA and RNA was performed for all 23 cases within the Molecular and Genomic Pathology Laboratory at using the to the A.D. et Full Text Full Text PDF PubMed Scopus Google Scholar were with and DNA and RNA and were performed using the The was used to and and testing using on a sequencing was on an to the and were with the for and number alterations. was to the for Molecular Pathology, American of Clinical and College of American for of in using a et 2017; Full Text Full Text PDF PubMed Scopus Google Scholar and were however, and were used for subsequent The of detection of the was at 3% The of for testing was number analysis was also performed as previously C. et Full Text Full Text PDF PubMed Scopus Google Scholar The of sequencing of each was by the and in sequencing established with a presence of a at was a number In an in sequencing established was as a number gene amplification was by the presence of of a as previously described and validated using fluorescence in situ hybridization C. et Full Text Full Text PDF PubMed Scopus Google et Scholar RNA expression and were evaluated using the and an The of detection of the RNA was than that the fusion are to a fusion RNA sequencing of cases 2, 8, 10, 11, and were using the RNA for to the generation and sequencing were performed on an using detection of gene sequencing were using and an for detection of et 2011; PubMed Scopus Google Scholar, D. et 2014; Scholar, et 2017; Scholar of sequencing for gene expression was performed as previously A.D. et Full Text Full Text PDF PubMed Scopus Google et PubMed Scopus Google Scholar gene expression the from sequencing were to human using and the number of to each gene was using and C. et 2009; PubMed Scopus Google Scholar, et 2011; PubMed Scopus Google Scholar, et 2014; 30: PubMed Scopus Google Scholar the the expression analysis was performed using the et 2010; PubMed Scopus Google Scholar The expression for target genes implicated in the growth factor kinase signaling were evaluated as previously A.D. et Full Text Full Text PDF PubMed Scopus Google Scholar with a of and a were as genes and used in pathway for cases was used to for MAML2 as previously et al.Am J Surg Pathol. 2010; 34: PubMed Scopus Google Scholar the MAML2 break-apart of an and that to the 5′ and 3′ of were to a paraffin-embedded were at 4 on for at and in were in and to in 10 acid was by an to proteins and The and were and were and was as well as a of the was by using a fluorescence and were by using a in for each with as of were from Figure MAPK fusion genes, such as those in FGFR2 and were identified by WTS of the each represents an of the gene with the The is of relative expression with expression and low the the of each gene are In addition to fusion genes, WTS detected novel fusion genes that have been implicated within the MAPK signaling pathway to RASGRF2 MAML2 and MAML3 Figure Figure WTS of identified an NCOA3-MAML2 fusion gene. The MAML2 rearrangement was confirmed using a break-apart that end of MAML2) and end of MAML2) with a In 8 harbored an ATP1B1-NRG1 fusion gene and demonstrated wild-type MAML2 to and Figure Figure WTS of intraductal papillary mucinous neoplasms n = and ITPNs (n = and key genomic alterations associated with ITPNs to signaling pathways. sequencing expression of genes implicated within multiple receptor kinase signaling pathways. However, ITPNs as compared with expression for genes in growth factor receptor kinase the identification of multiple signaling and based on previous studies, the key genomic alterations that ITPNs to the MAPK signaling pathway, which is to a of such as cell and high-grade Figure