title: Reporting tumor molecular heterogeneity in histopathological diagnosis.
abstract: BACKGROUND
  Detection of molecular tumor heterogeneity has become of paramount importance with the advent of targeted therapies. Analysis for detection should be comprehensive, timely and based on routinely available tumor samples.
  
  
  OBJECTIVE
  To evaluate the diagnostic potential of targeted multigene next-generation sequencing (TM-NGS) in characterizing gastrointestinal cancer molecular heterogeneity.
  
  
  METHODS
  35 gastrointestinal tract tumors, five of each intestinal type gastric carcinomas, pancreatic ductal adenocarcinomas, pancreatic intraductal papillary mucinous neoplasms, ampulla of Vater carcinomas, hepatocellular carcinomas, cholangiocarcinomas, pancreatic solid pseudopapillary tumors were assessed for mutations in 46 cancer-associated genes, using Ion Torrent semiconductor-based TM-NGS. One ampulla of Vater carcinoma cell line and one hepatic carcinosarcoma served to assess assay sensitivity. TP53, PIK3CA, KRAS, and BRAF mutations were validated by conventional Sanger sequencing.
  
  
  RESULTS
  TM-NGS yielded overlapping results on matched fresh-frozen and formalin-fixed paraffin-embedded (FFPE) tissues, with a mutation detection limit of 1% for fresh-frozen high molecular weight DNA and 2% for FFPE partially degraded DNA. At least one somatic mutation was observed in all tumors tested; multiple alterations were detected in 20/35 (57%) tumors. Seven cancers displayed significant differences in allelic frequencies for distinct mutations, indicating the presence of intratumor molecular heterogeneity; this was confirmed on selected samples by immunohistochemistry of p53 and Smad4, showing concordance with mutational analysis.
  
  
  CONCLUSIONS
  TM-NGS is able to detect and quantitate multiple gene alterations from limited amounts of DNA, moving one step closer to a next-generation histopathologic diagnosis that integrates morphologic, immunophenotypic, and multigene mutational analysis on routinely processed tissues, essential for personalized cancer therapy.
authors.affiliation: Applied Research on Cancer Network (ARC-NET) and Department of Pathology and Diagnostics, University and Hospital Trust of Verona, Verona, Italy.
authors.forename: Andrea
authors.initials: A
authors.lastname: Mafficini
authors.affiliation: Applied Research on Cancer Network (ARC-NET) and Department of Pathology and Diagnostics, University and Hospital Trust of Verona, Verona, Italy.
authors.forename: Eliana
authors.initials: E
authors.lastname: Amato
authors.affiliation: Applied Research on Cancer Network (ARC-NET) and Department of Pathology and Diagnostics, University and Hospital Trust of Verona, Verona, Italy.
authors.forename: Matteo
authors.initials: M
authors.lastname: Fassan
authors.affiliation: Applied Research on Cancer Network (ARC-NET) and Department of Pathology and Diagnostics, University and Hospital Trust of Verona, Verona, Italy.
authors.forename: Michele
authors.initials: M
authors.lastname: Simbolo
authors.affiliation: Applied Research on Cancer Network (ARC-NET) and Department of Pathology and Diagnostics, University and Hospital Trust of Verona, Verona, Italy; Department of Surgery, University and Hospital Trust of Verona, Verona, Italy.
authors.forename: Davide
authors.initials: D
authors.lastname: Antonello
authors.affiliation: Applied Research on Cancer Network (ARC-NET) and Department of Pathology and Diagnostics, University and Hospital Trust of Verona, Verona, Italy.
authors.forename: Caterina
authors.initials: C
authors.lastname: Vicentini
authors.affiliation: Applied Research on Cancer Network (ARC-NET) and Department of Pathology and Diagnostics, University and Hospital Trust of Verona, Verona, Italy.
authors.forename: Maria
authors.initials: M
authors.lastname: Scardoni
authors.affiliation: Applied Research on Cancer Network (ARC-NET) and Department of Pathology and Diagnostics, University and Hospital Trust of Verona, Verona, Italy.
authors.forename: Samantha
authors.initials: S
authors.lastname: Bersani
authors.affiliation: Applied Research on Cancer Network (ARC-NET) and Department of Pathology and Diagnostics, University and Hospital Trust of Verona, Verona, Italy.
authors.forename: Marisa
authors.initials: M
authors.lastname: Gottardi
authors.affiliation: Applied Research on Cancer Network (ARC-NET) and Department of Pathology and Diagnostics, University and Hospital Trust of Verona, Verona, Italy.
authors.forename: Borislav
authors.initials: B
authors.lastname: Rusev
authors.affiliation: Applied Research on Cancer Network (ARC-NET) and Department of Pathology and Diagnostics, University and Hospital Trust of Verona, Verona, Italy; Department of Surgery, University and Hospital Trust of Verona, Verona, Italy.
authors.forename: Giorgio
authors.initials: G
authors.lastname: Malpeli
authors.affiliation: Applied Research on Cancer Network (ARC-NET) and Department of Pathology and Diagnostics, University and Hospital Trust of Verona, Verona, Italy.
authors.forename: Vincenzo
authors.initials: V
authors.lastname: Corbo
authors.affiliation: Applied Research on Cancer Network (ARC-NET) and Department of Pathology and Diagnostics, University and Hospital Trust of Verona, Verona, Italy.
authors.forename: Stefano
authors.initials: S
authors.lastname: Barbi
authors.affiliation: Applied Research on Cancer Network (ARC-NET) and Department of Pathology and Diagnostics, University and Hospital Trust of Verona, Verona, Italy.
authors.forename: Katarzyna O
authors.initials: KO
authors.lastname: Sikora
authors.affiliation: Applied Research on Cancer Network (ARC-NET) and Department of Pathology and Diagnostics, University and Hospital Trust of Verona, Verona, Italy.
authors.forename: Rita T
authors.initials: RT
authors.lastname: Lawlor
authors.affiliation: Department of Medicine, Oncology Unit, University and Hospital Trust of Verona, Verona, Italy.
authors.forename: Giampaolo
authors.initials: G
authors.lastname: Tortora
authors.affiliation: Applied Research on Cancer Network (ARC-NET) and Department of Pathology and Diagnostics, University and Hospital Trust of Verona, Verona, Italy.
authors.forename: Aldo
authors.initials: A
authors.lastname: Scarpa
mesh_terms: D001483:Base Sequence; D058534:Class I Phosphatidylinositol 3-Kinases; D004252:DNA Mutational Analysis / Q000379:methods*; D059014:High-Throughput Nucleotide Sequencing / Q000379:methods*; D006801:Humans; D009369:Neoplasms / Q000235:genetics* / Q000473:pathology*; D016612:Paraffin Embedding; D019869:Phosphatidylinositol 3-Kinases / Q000235:genetics; D011518:Proto-Oncogene Proteins / Q000235:genetics; D048493:Proto-Oncogene Proteins B-raf / Q000235:genetics; D016283:Proto-Oncogene Proteins p21(ras); D016707:Tissue Fixation; D016159:Tumor Suppressor Protein p53 / Q000235:genetics; D018631:ras Proteins / Q000235:genetics
chemical_list: C117307:KRAS protein, human; D011518:Proto-Oncogene Proteins; D016159:Tumor Suppressor Protein p53; D019869:Phosphatidylinositol 3-Kinases; D058534:Class I Phosphatidylinositol 3-Kinases; C484760:PIK3CA protein, human; D048493:Proto-Oncogene Proteins B-raf; D016283:Proto-Oncogene Proteins p21(ras); D018631:ras Proteins