Abstract

Solid-pseudopapillary tumors (SPTs) are unusual pancreatic neoplasms of low malignant potential that most frequently affect young women. Genetic events contributing to the development of SPTs are unknown. Whereas the more common ductal adenocarcinomas of the pancreas essentially never harbor β-catenin or APC gene mutations, we have recently identified alterations of the APC/β-catenin pathway in other nonductal pancreatic neoplasms including pancreatoblastomas and acinar cell carcinomas. We analyzed a series of 20 SPTs for somatic alterations of the APC/β-catenin pathway using immunohistochemistry for β-catenin protein accumulation, direct DNA sequencing of β-catenin exon 3, and direct DNA sequencing of the mutation cluster region in exon 15 of the APC gene in those SPTs that did not harbor β-catenin mutations. Immunohistochemical labeling for cyclin D1 was performed to evaluate the overexpression of this cell-cycle protein as one of the putative downstream effectors of β-catenin dysregulation. In addition, we analyzed the SPTs for genetic alterations commonly found in pancreatic ductal adenocarcinomas, including mutations in the K-ras oncogene and p53 and DPC4 tumor suppressor genes, using direct DNA sequencing of K-ras and immunostaining for p53 and Dpc4. Almost all SPTs harbored alterations in the APC/β-catenin pathway. Nuclear accumulation of β-catenin protein was present in 95% (19 of 20), and activating β-catenin oncogene mutations were identified in 90% (18 of 20) of the SPTs. Seventy-four percent (14 of 19) showed overexpression of cyclin D1, ranging from 10 to 70% of tumor nuclei. In contrast, no K-ras mutations were present in any of the 20 SPTs, and Dpc4 expression was intact in all 16 SPTs for which immunohistochemical labeling was successful. Overexpression of p53 was limited to only 3 of 19 (15.8%) SPTs. These results emphasize the two distinct, divergent genetic pathways of neoplastic progression in pancreatic ductal and nonductal neoplasms. Solid-pseudopapillary tumors (SPTs) are unusual pancreatic neoplasms of low malignant potential that most frequently affect young women. Genetic events contributing to the development of SPTs are unknown. Whereas the more common ductal adenocarcinomas of the pancreas essentially never harbor β-catenin or APC gene mutations, we have recently identified alterations of the APC/β-catenin pathway in other nonductal pancreatic neoplasms including pancreatoblastomas and acinar cell carcinomas. We analyzed a series of 20 SPTs for somatic alterations of the APC/β-catenin pathway using immunohistochemistry for β-catenin protein accumulation, direct DNA sequencing of β-catenin exon 3, and direct DNA sequencing of the mutation cluster region in exon 15 of the APC gene in those SPTs that did not harbor β-catenin mutations. Immunohistochemical labeling for cyclin D1 was performed to evaluate the overexpression of this cell-cycle protein as one of the putative downstream effectors of β-catenin dysregulation. In addition, we analyzed the SPTs for genetic alterations commonly found in pancreatic ductal adenocarcinomas, including mutations in the K-ras oncogene and p53 and DPC4 tumor suppressor genes, using direct DNA sequencing of K-ras and immunostaining for p53 and Dpc4. Almost all SPTs harbored alterations in the APC/β-catenin pathway. Nuclear accumulation of β-catenin protein was present in 95% (19 of 20), and activating β-catenin oncogene mutations were identified in 90% (18 of 20) of the SPTs. Seventy-four percent (14 of 19) showed overexpression of cyclin D1, ranging from 10 to 70% of tumor nuclei. In contrast, no K-ras mutations were present in any of the 20 SPTs, and Dpc4 expression was intact in all 16 SPTs for which immunohistochemical labeling was successful. Overexpression of p53 was limited to only 3 of 19 (15.8%) SPTs. These results emphasize the two distinct, divergent genetic pathways of neoplastic progression in pancreatic ductal and nonductal neoplasms. Solid-pseudopapillary tumors of the pancreas (SPTs) are uncommon tumors, constituting only ∼1% of pancreatic neoplasms.1Klimstra DS Wenig BM Heffess CS Solid-pseudopapillary tumor of the pancreas: a typically cystic carcinoma of low malignant potential.Semin Diagn Pathol. 2000; 17: 66-80PubMed Google Scholar SPTs are histologically, clinically, and prognostically quite distinct from the more common pancreatic ductal adenocarcinomas. In contrast to the infiltrative growth and almost invariably lethal behavior of ductal adenocarcinomas among older individuals, SPTs are neoplasms of young women and are frequently grossly sharply circumscribed and cystic lesions (although their microscopic margins may be indistinct). Most importantly, despite their often large size (mean of >10 cm at presentation in one study),2Mao C Guvendi M Domenico DR Kim K Thomford NR Howard JM Papillary cystic and solid tumors of the pancreas: a pancreatic embryonic tumor? Studies of three cases and cumulative review of the world's literature.Surgery. 1995; 118: 821-828Abstract Full Text PDF PubMed Scopus (252) Google Scholar the vast majority of SPTs are indolent neoplasms. They are confined to the pancreas in 85% of patients, and even the 10 to 15% of patients with liver or peritoneal metastases from SPTs commonly enjoy long-term survival.2Mao C Guvendi M Domenico DR Kim K Thomford NR Howard JM Papillary cystic and solid tumors of the pancreas: a pancreatic embryonic tumor? Studies of three cases and cumulative review of the world's literature.Surgery. 1995; 118: 821-828Abstract Full Text PDF PubMed Scopus (252) Google Scholar, 3Horisawa M Niinomi N Sato T Yokoi S Oda K Ichikawa M Hayakawa S Frantz's tumor (solid and cystic tumor of the pancreas) with liver metastasis: successful treatment and long-term follow-up.J Pediatr Surg. 1995; 30: 724-726Abstract Full Text PDF PubMed Scopus (87) Google Scholar, 4Rebhandl W Felberbauer FX Puig S Paya K Hochschorner S Barlan M Horcher E Solid-pseudopapillary tumor of the pancreas (Frantz tumor) in children: report of four cases and review of the literature.J Surg Oncol. 2001; 76: 289-296Crossref PubMed Scopus (143) Google Scholar, 5Ogawa T Isaji S Okamura K Noguchi T Mizumoto R Ishihara A A case of radical resection for solid-cystic tumor of the pancreas with widespread metastases in the liver and greater omentum.Am J Gastroenterol. 1993; 88: 1436-1439PubMed Google Scholar, 6Saiura A Umekita N Matsui Y Maeshiro T Miyamoto S Kitamura M Wakikawa A Successful surgical resection of solid cystic tumor of the pancreas with multiple liver metastases and a tumor thrombus in the portal vein.Hepatogastroenterology. 2000; 47: 887-889PubMed Google ScholarThe histopathological appearance of SPTs is distinctive among the primary pancreatic neoplasms.1Klimstra DS Wenig BM Heffess CS Solid-pseudopapillary tumor of the pancreas: a typically cystic carcinoma of low malignant potential.Semin Diagn Pathol. 2000; 17: 66-80PubMed Google Scholar The neoplastic epithelial cells are uniform, polygonal, and discohesive in nature. Smaller SPTs may be primarily arranged in solid sheets with a rich microvasculature (Figure 1A). Frequent degenerative changes in larger tumors, however, lend a characteristic pseudopapillary pattern because of residual epithelial cells that form perivascular rosettes (Figure 1B). SPTs are known to consistently express vimentin,7Pettinato G Manivel JC Ravetto C Terracciano LM Gould EW di Tuoro A Jaszcz W Albores-Saavedra J Papillary cystic tumor of the pancreas. 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Clinical, histochemical, and electron microscopic features in ten cases.Cancer. 1991; 67: 1635-1641Crossref PubMed Scopus (125) Google Scholar progesterone receptors,10Kosmahl M Seada LS Janig U Harms D Kloppel G Solid-pseudopapillary tumor of the pancreas: its origin revisited.Virchows Arch. 2000; 436: 473-480Crossref PubMed Scopus (278) Google Scholar and more recently, CD10.9Notohara K Hamazaki S Tsukayama C Nakamoto S Kawabata K Mizobuchi K Sakamoto K Okada S Solid-pseudopapillary tumor of the pancreas: immunohistochemical localization of neuroendocrine markers and CD10.Am J Surg Pathol. 2000; 24: 1361-1371Crossref PubMed Scopus (223) Google Scholar However, attempts to discern a cell of origin or even a line of cellular differentiation for SPTs based on these results have been unrevealing.10Kosmahl M Seada LS Janig U Harms D Kloppel G Solid-pseudopapillary tumor of the pancreas: its origin revisited.Virchows Arch. 2000; 436: 473-480Crossref PubMed Scopus (278) Google Scholar, 12Balercia G Zamboni G Bogina G Mariuzzi GM Solid-cystic tumor of the pancreas. 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These genes are frequently involved in the pathogenesis of pancreatic ductal adenocarcinomas. Not surprisingly, given the marked clinicopathological differences between SPTs and ductal adenocarcinomas, alterations in these genes have not been detected in SPTs.18Flejou JF Boulange B Bernades P Belghiti J Henin D p53 protein expression and DNA ploidy in cystic tumors of the pancreas.Pancreas. 1996; 13: 247-252Crossref PubMed Scopus (42) Google Scholar, 19Lee WY Tzeng CC Chen RM Tsao CJ Tseng JY Jin YT Papillary cystic tumors of the pancreas: assessment of malignant potential by analysis of progesterone receptor, flow cytometry, and ras oncogene mutation.Anticancer Res. 1997; 17: 2587-2591PubMed Google Scholar, 23Bartsch D Bastian D Barth P Schudy A Nies C Kisker O Wagner HJ Rothmund M K-ras oncogene mutations indicate malignancy in cystic tumors of the pancreas.Ann Surg. 1998; 228: 79-86Crossref PubMed Scopus (85) Google Scholar, 24Yamaue H Tanimura H Shono Y Onishi H Tani M Yamoto H Kinoshita H Uchiyama K Solid and cystic tumor of the pancreas: clinicopathologic and genetic studies of four cases.Int J Pancreatol. 2000; 27: 69-76Crossref PubMed Google Scholar, 25Muller-Hocker J Zietz CH Sendelhofert A Deregulated expression of cell cycle-associated proteins in solid pseudopapillary tumor of the pancreas.Mod Pathol. 2001; 14: 47-53Crossref PubMed Scopus (43) Google Scholar, 26Lam KY Lo CY Fan ST Pancreatic solid-cystic-papillary tumor: clinicopathologic features in eight patients from Hong Kong and review of the literature.World J Surg. 1999; 23: 1045-1050Crossref PubMed Scopus (233) Google Scholar, 27Moore PS Orlandini S Zamboni G Capelli P Rigaud G Falconi M Bassi C Lemoine NR Scarpa A Pancreatic tumours: molecular pathways implicated in ductal cancer are involved in ampullary but not in exocrine nonductal or endocrine tumorigenesis.Br J Cancer. 2001; 84: 253-262Crossref PubMed Scopus (170) Google ScholarIn contrast to pancreatic ductal adenocarcinomas in which β-catenin or APC gene mutations are essentially never present, we have recently identified somatic alterations of the APC/β-catenin pathway in other pancreatic nonductal neoplasms including pancreatoblastomas and acinar cell carcinomas.28Abraham SC Wu TT Klimstra DS Finn L Lee JH Yeo CJ Cameron JL Hruban RH Distinctive molecular genetic alterations in sporadic and familial adenomatous polyposis-associated pancreatoblastomas: frequent alterations in the APC/β-catenin pathway and chromosome 11p.Am J Pathol. 2001; 159: 1619-1627Abstract Full Text Full Text PDF PubMed Scopus (211) Google Scholar, 29Abraham SC Wu TT Hruban RH Lee JH Yeo CJ Conlon K Brennan M Cameron JL Klimstra DS Genetic and immunohistochemical analysis of pancreatic acinar cell carcinoma: frequent allelic loss on chromosome 11p and alterations in the APC/β-catenin pathway.Am J Pathol. 2002; 160: 953-962Abstract Full Text Full Text PDF PubMed Scopus (219) Google Scholar We therefore undertook a molecular characterization of a series of 20 SPTs for alterations in the APC/β-catenin pathway and for cyclin D1 protein overexpression, a putative downstream effector of β-catenin dysregulation.30Tetsu O McCormick F β-catenin regulates expression of cyclin D1 in colon carcinoma cells.Nature. 1999; 398: 422-426Crossref PubMed Scopus (3238) Google Scholar, 31Mann B Gelos M Siedow A Hanski ML Gratchev A Ilyas M Bodmer WF Moyer MP Riecken EO Buhr HJ Hanski C Target genes of β-catenin-T cell-factor/lymphoid-enhancer-factor signaling in human colorectal carcinomas.Proc Natl Acad Sci USA. 1999; 96: 1603-1608Crossref PubMed Scopus (720) Google Scholar, 32Sellin JH Umar S Xiao J Morris AP Increased β-catenin expression and nuclear translocation accompany cellular hyperproliferation in vivo.Cancer Res. 2001; 61: 2899-2906PubMed Google Scholar, 33Takayasu H Horie H Hiyama E Matsunaga T Hayashi Y Watanabe Y Suita S Kaneko M Sasaki F Hashizume K Ozaki T Furuuchi K Tada M Ohnuma N Nakagawara A Frequent deletions and mutations of the beta-catenin gene are associated with overexpression of cyclin D1 and fibronectin and poorly differentiated histology in childhood hepatoblastoma.Clin Cancer Res. 2001; 7: 901-908PubMed Google Scholar In addition, we analyzed the SPTs for alterations in the K-ras oncogene and p53 and DPC4 tumor suppressor genes that are known to characterize pancreatic ductal adenocarcinomas.Materials and MethodsCase SelectionThe study population consisted of 20 patients with pancreatic SPTs who either underwent surgical resection or whose surgical material was seen in consultation at The Johns Hopkins Hospital (9 cases) or Memorial Sloan-Kettering Cancer Center (11 cases) between 1989 and 2000. Clinical information including patient age, gender, tumor size, tumor location in the pancreas, and presence or absence of metastatic disease was collected from patient charts or the computerized patient files.Immunohistochemistry for β-Catenin, p53, and Dpc4Immunohistochemical labeling using diaminobenzidine as the chromogen was performed on the Techmate 1000 automatic labeling system (BioTek Solutions, Tucson, AZ). Deparaffinized sections of formalin-fixed tissue at 5 μm thickness were labeled with β-catenin antibody (1:500 dilution, mouse monoclonal; Becton Dickinson Transduction Laboratories, Lexington, KY), cyclin D1 antibody (1:50 dilution, rabbit polyclonal; Oncogene Research Products, San Diego, CA), p53 antibody (1:100 dilution, mouse monoclonal clone D07; DAKO, Carpinteria, CA), and Dpc4 antibody (1:100 dilution, monoclonal clone B8; Santa Cruz Biotechnology, Santa Cruz, CA). Heat-induced antigen retrieval using steam for 20 minutes at 80°C was used before incubation with all four antibodies.For β-catenin, immunohistochemical labeling was evaluated for the presence of nuclear, cytoplasmic, and membranous β-catenin accumulation in the SPTs and surrounding nonneoplastic pancreas, if present. Nuclear and cytoplasmic accumulation of β-catenin in SPTs was graded according to the percentage of neoplastic cells with strong immunolabeling. For cyclin D1, the percentage of moderately or strongly positive tumor nuclei was evaluated. For p53, the percentage of positively labeled nuclei was recorded; we considered strong nuclear labeling in ≥30% of neoplastic cells as the cutoff for positivity.34Baas IO Mulder JW Offerhaus GJ Vogelstein B Hamilton SR An evaluation of six antibodies for immunohistochemistry of mutant p53 gene product in archival colorectal neoplasms.J Pathol. 1994; 172: 5-12Crossref PubMed Scopus (528) Google Scholar For Dpc4, SPTs were classified as showing intact Dpc4 protein expression if they showed the normal pattern of strong, diffuse cytoplasmic labeling and labeling of scattered nuclei. They were classified as showing loss of normal Dpc4 expression only if they showed a complete loss of cytoplasmic and nuclear Dpc4 labeling.35Wilentz RE Su GH Dai JL Sparks AB Argani P Sohn TA Yeo CJ Kern SE Rhuban RH Immunohistochemical labeling for Dpc4 mirrors genetic status in pancreatic adenocarcinomas: a new marker of DPC4 inactivation.Am J Pathol. 2000; 156: 37-43Abstract Full Text Full Text PDF PubMed Scopus (286) Google ScholarDNA ExtractionMicrodissection of SPTs for DNA extraction was performed from formalin-fixed, paraffin-embedded tissues. A 271/2-guage-needle tip was used for microdissection of routinely processed 5-μm hematoxylin and eosin-stained slides under a low-power (×4) objective. Genomic DNA was extracted as described previously.36Moskaluk CA Kern SE Microdissection and polymerase chain reaction amplification of genomic DNA from histological tissue sections.Am J Pathol. 1997; 150: 1547-1552PubMed Google Scholar Corresponding normal control DNA was available in 17 cases and was extracted from adjacent nonneoplastic pancreatic acini or stroma in 16 cases and from adjacent duodenum in 1 case.Mutation Analysis of the β-Catenin and APC GenesGenomic DNA from each SPT and normal tissue (if present) was amplified by polymerase chain reaction (PCR) using the primer pair: 5′-ATGGAACCAGACAGAAAAGC-3′ (sense) and 5′-GCTACTTGTTCTGAGTGAAG-3′ (anti-sense). These amplified a 200-bp fragment of exon 3 of the β-catenin gene that encompasses the region for GSK-3β phosphorylation. PCR reactions were performed under standard conditions in a 50-μl volume containing 38 μl of Platinum PCR SuperMix (Life Technologies, Rockville, MD), 5 μl of both 5′ and 3′ oligonucleotides (final concentration of 1 μmol/L), and 2 μl (∼50 ng) of genomic DNA. PCR conditions consisted of an initial denaturation at 94°C for 3 minutes, 40 cycles of 94°C for 1 minute, 58°C for 1 minute, and 72°C for 2 minutes, and a final extension at 72°C for 7 minutes. PCR products were purified with spin columns using QIAquick PCR purification kit (Qiagen, Inc., Valencia, CA) before sequencing. Automated sequencing of purified PCR products was performed on an ABI Prism 3700 DNA Analyzer (Applied Biosystems, Inc., Foster City, CA) using the internal primers: 5′-AAAGCGGCTGTTAGTCACTGG-3′ (sense) and 5′-CCTGTTCCCACTCATACAGG-3′ (anti-sense), and the resulting sequence data were analyzed with the Sequencher analysis program (Gene Codes, Ann Arbor, MI). All mutations were verified in both sense and anti-sense directions on independent PCR products.Direct sequencing for APC gene mutations was attempted for SPTs that did not contain identifiable β-catenin mutations. Four sets of oligonucleotide primers (A1: 5′-CAGACTTATTGTGTAGAAGA-3′ and A2: 5′-CTCCTGAAGAAAATTCAACA-3′ for codons 1260–1359; B1: 5′-AGGGTTCTAGTTTATCTTCA-3′ and B2: 5′-TCTGCTTGGTGGCATGGTTT-3′ for codons 1339–1436; C1: 5′-GGCATTATAAGCCCCAGTGA-3′ and C2: 5′-AAATGG-CTCATCGAGGCTCA-3′ for codons 1417–1516; D1: 5′-ACTCCAGATGGATTTTCTTG-3′ and D2: 5′-GGCTGGCT-TTTTTGCTTTAC-3′ for codons 1497–1596) were used to amplify the mutation cluster region of the APC gene.37Yashima K Nakamori S Murakami Y Yamaguchi A Hayashi K Ishikawa O Konishi Y Sekiya T Mutations of the adenomatous polyposis coli gene in the mutation cluster region: comparison of human pancreatic and colorectal cancers.Int J Cancer. 1994; 59: 43-47Crossref PubMed Scopus (67) Google Scholar PCR reactions were performed in 50-μl volumes using the reaction mixture described above. PCR conditions consisted of an initial denaturation step of 94°C for 3 minutes, 40 cycles (94°C for 1 minute, 55°C for 1 minute, and 68°C for 1.5 minutes for APC-B, APC-C, and APC-D primer pairs and 94°C for 1 minute, 52°C for 1 minute, and 68°C for 1.5 minutes for APC-A), followed by a final extension at 72°C for 7 minutes.Mutation Analysis of the K-ras OncogeneThe oligonucleotide primers 5′-GAGAATTCATGACTGAATATAAACTTGT-3′ (sense) and 5′-TCGAATTCCTCTATTGTTGGATCATATTCG-3′ (anti-sense) were used to amplify a region in exon 1 of the K-ras gene that includes codons 12 and 13. PCR reactions were performed in 50-μl volumes using the reaction mixture described above, with an initial denaturation at 94°C for 3 minutes, 40 cycles of 94°C for 1 minute, 50°C for 1 minute, and 72°C for 1 minute, and a final extension at 72°C for 7 minutes. PCR products were purified and sequenced as described above using the internal primer 5′-ATTCGTCCACAAAATGAT-3′.ResultsA summary of the clinicopathological features of the 20 SPTs (designated S1 to S20) is shown in Table 1, and the corresponding molecular findings are shown in Table 2.Table 1Clinicopathological Features of Pancreatic Solid-Psuedopapillary TumorsCaseAge/sexTumor sizeLocation in pancreasNuclear pleomorphismInvasion of adjacent structuresMetastasesS157/M6.0 cmTail−−−S235/F3.5 cmBody−−−S342/F2.0 cmHead−−−S425/F7.5 cmBody/tail−−−S538/F4.7 cmBody/tail−−−S637/F2.5 cmBody+ (Focal)+ (Peripancreatic fat)−S751/F3.5 cmUncinate−−−S857/F5.2 cmHead−+ (Mesocolon; duodenum; peripancreatic fat)−S975/F5.0 cmHead−−−S1056/F4.0 cmDistal−−+ (Liver)*This patient died of disease 10 years after presentation.S1118/F7.5 cmHead−−−S12N/AN/AN/A−N/AN/AS1313/M5.5 cmN/A−−−S1449/F5.5 cmDistal−+ (Vascular, perineural)−S1538/F1.0 cmDistal−+ (Perineural)−S1638/M>2 cmTail−+ (Capsular vessels)−S1742/F2.5 cmDistal−−−S1826/F11.5 cmHead−−−S1942/F3.0 cmDistal−−−S2027/F4.5 cmHead−−−N/A, information was not available.* This patient died of disease 10 years after presentation. Open table in a new tab Table 2Genetic Alterations in Pancreatic Solid-Pseudopapillary TumorsCaseβ-catenin mutationNuclear β-catenin, %Nuclear cyclin D1, %K-ras mutationp53 accumulationDpc4 lossS1G34V>9050Wild-type−IntactS2S37F>9030Wild-type−IntactS3D32Y>9040Wild-type−IntactS4G34V>9050Wild-type−IntactS5S37F>9070Wild-type−IntactS6G34V>9010Wild-type+ (Patchy)IntactS7S33A>9020Wild-type−IntactS8S37F>9010Wild-type−IntactS9D32V>9010Wild-type−IntactS10Wild-type>90−Wild-type−IntactS11D32N20−Wild-type−N/AS12S37F30−Wild-type−IntactS13S37F>9015Wild-type−IntactS14Wild-type>90−Wild-type40%IntactS15G34R>9010Wild-type−IntactS16S37F>9030Wild-type−IntactS17D32Y>9030Wild-type−N/AS18D32N>90−Wild-type−IntactS19D32Y>9010Wild-type+ (Patchy)N/AS20G34RN/AN/AWild-typeN/AN/ALocations of somatic mutations in β-catenin are shown by codon.Nuclear β-catenin, cyclin D1, and p53 accumulation were evaluated based on the percentage of labeled tumor cell nuclei. Open table in a new tab Clinicopathological CharacteristicsClinical information was available in 19 patients with SPTs. Sixteen (84.2%) patients were female and three (15.8%) were male. They ranged from 13 to 75 years with a mean patient age of 40.3 years. The SPTs were located throughout the pancreas without a particular site predilection. Mean size of the SPTs was 4.6 cm, although there was a wide variation in tumor size, ranging from 1 cm to 11.5 cm. Histopathologically, the 20 SPTs showed uniformly small, regular nuclei with a focal area of nuclear pleomorphism in only one (5%) of the SPTs. Five of the 19 SPTs (26.3%) for which pathological staging was available showed tumor infiltration either into vascular/perineural spaces (three SPTs) or into the adjacent bowel wall and/or peripancreatic fat (two SPTs). However, metastatic disease was present in only one patient (S10, with metastases to the liver), who subsequently died of disease 10 years after initial presentation.Mutations in the β-Catenin GeneActivating mutations in exon