title: RASSF1 tumor suppressor gene in pancreatic ductal adenocarcinoma: correlation of expression, chromosomal status and epigenetic changes.
abstract: BACKGROUND
  The Ras Association Domain Family Member 1 (RASSF1) is one of the most frequently reported methylation-inactivated tumor suppressor genes in primary pancreatic ductal adenocarcinomas (PDAC). Limited information is still available about the impact of RASSF1 gene silencing on the expression of its different isoforms in neoplastic cells.
  
  
  METHODS
  A series of 96 primary PDAC, with known clinico-pathological parameters, was tested for RASSF1 methylation status by methylation-specific PCR, RASSF1 locus copy number alterations by fluorescence in situ hybridization, and Rassf1a protein expression by immunohistochemistry. A further series of 14 xenografted primary PDAC and 8 PDAC-derived cell lines were tested to obtain a detailed methylation mapping of CpG islands A and C of the RASSF1 locus by pyrosequencing and to evaluate the expression of Rassf1 variants by qRT-PCR.
  
  
  RESULTS
  Methylation of CpG island A of the RASSF1 gene was observed in 35% of the tumors and allelic loss of RASSF1 locus was seen in 30 disomic and in 20 polysomic cases (52%). Rassf1a immunohistochemical expression was downregulated in half of primary PDAC, and this downregulation was neither correlated with methylation of RASSF1 promoter nor with RASSF1 copy number alterations. RASSF1 status did not influence patients' prognosis. The expression of the seven RASSF1 isoforms in xenografts and cell lines showed that RASSF1A, RASSF1B, and RASSF1C isoforms were present in all xenografts and cell lines, whereas RASSF1D, RASSF1E, and RASSF1F isoforms were variably expressed among samples. RASSF1G was never expressed in either xenografts or cell lines. The variable expression of RASSF1 isoforms in PDAC xenografts and cell lines was not dependent on RASSF1 methylation status of CpG islands A and C.
  
  
  CONCLUSIONS
  RASSF1 alterations occurring in PDAC mainly consist in variations of expression of the different isoforms. Different genetic mechanisms seem to contribute to RASSF1 deregulation in this setting, but RASSF1 methylation does not seem to substantially affect RASSF1 isoforms expression.
authors.affiliation: ARC-NET Centre for Applied Research on Cancer, Department of Pathology and Diagnostics, The Hospital and University of Verona, Verona, Italy. eliana.amato@gmail.com.
authors.forename: Eliana
authors.initials: E
authors.lastname: Amato
authors.affiliation: ARC-NET Centre for Applied Research on Cancer, Department of Pathology and Diagnostics, The Hospital and University of Verona, Verona, Italy. stefano.barbi@univr.it.
authors.forename: Stefano
authors.initials: S
authors.lastname: Barbi
authors.affiliation: ARC-NET Centre for Applied Research on Cancer, Department of Pathology and Diagnostics, The Hospital and University of Verona, Verona, Italy. matteo.fassan@gmail.com.
authors.forename: Matteo
authors.initials: M
authors.lastname: Fassan
authors.affiliation: ARC-NET Centre for Applied Research on Cancer, Department of Pathology and Diagnostics, The Hospital and University of Verona, Verona, Italy. claudio.luchini@univr.it.|Department of Pathology, The Hospital and University of Verona, Verona, Italy. claudio.luchini@univr.it.
authors.forename: Claudio
authors.initials: C
authors.lastname: Luchini
authors.affiliation: ARC-NET Centre for Applied Research on Cancer, Department of Pathology and Diagnostics, The Hospital and University of Verona, Verona, Italy. caterinavicentini@hotmail.it.
authors.forename: Caterina
authors.initials: C
authors.lastname: Vicentini
authors.affiliation: ARC-NET Centre for Applied Research on Cancer, Department of Pathology and Diagnostics, The Hospital and University of Verona, Verona, Italy. matteo.brunelli@univr.it.
authors.forename: Matteo
authors.initials: M
authors.lastname: Brunelli
authors.affiliation: Department of Surgery and Oncology, The Hospital and University of Verona, Verona, Italy. giuseppe.malleo@ospedaleuniverona.it.
authors.forename: Giuseppe
authors.initials: G
authors.lastname: Malleo
authors.affiliation: ARC-NET Centre for Applied Research on Cancer, Department of Pathology and Diagnostics, The Hospital and University of Verona, Verona, Italy. aldo.scarpa@univr.it.|Department of Pathology, The Hospital and University of Verona, Verona, Italy. aldo.scarpa@univr.it.
authors.forename: Aldo
authors.initials: A
authors.lastname: Scarpa
authors.affiliation: ARC-NET Centre for Applied Research on Cancer, Department of Pathology and Diagnostics, The Hospital and University of Verona, Verona, Italy. giorgio.malpeli@univr.it.|Department of Surgery and Oncology, The Hospital and University of Verona, Verona, Italy. giorgio.malpeli@univr.it.
authors.forename: Giorgio
authors.initials: G
authors.lastname: Malpeli
mesh_terms: D000230:Adenocarcinoma / Q000235:genetics* / Q000473:pathology; D000368:Aged; D000818:Animals; D021441:Carcinoma, Pancreatic Ductal / Q000235:genetics* / Q000473:pathology; D018899:CpG Islands / Q000235:genetics; D019175:DNA Methylation / Q000235:genetics*; D044127:Epigenesis, Genetic; D005260:Female; D015972:Gene Expression Regulation, Neoplastic; D006801:Humans; D017404:In Situ Hybridization, Fluorescence; D019656:Loss of Heterozygosity / Q000235:genetics; D008297:Male; D051379:Mice; D008875:Middle Aged; D011401:Promoter Regions, Genetic; D025521:Tumor Suppressor Proteins / Q000096:biosynthesis / Q000235:genetics*; D023041:Xenograft Model Antitumor Assays
chemical_list: C411762:RASSF1 protein, human; D025521:Tumor Suppressor Proteins