title: Targeting EGFR/HER2 pathways enhances the antiproliferative effect of gemcitabine in biliary tract and gallbladder carcinomas.
abstract: BACKGROUND
  Advanced biliary tract carcinomas (BTCs) have poor prognosis and limited therapeutic options. Therefore, it is crucial to combine standard therapies with molecular targeting. In this study EGFR, HER2, and their molecular transducers were analysed in terms of mutations, amplifications and over-expression in a BTC case series. Furthermore, we tested the efficacy of drugs targeting these molecules, as single agents or in combination with gemcitabine, the standard therapeutic agent against BTC.
  
  
  METHODS
  Immunohistochemistry, FISH and mutational analysis were performed on 49 BTC samples of intrahepatic (ICCs), extrahepatic (ECCs), and gallbladder (GBCs) origin. The effect on cell proliferation of different EGFR/HER2 pathway inhibitors as single agents or in combination with gemcitabine was investigated on BTC cell lines. Western blot analyses were performed to investigate molecular mechanisms of targeted drugs.
  
  
  RESULTS
  EGFR is expressed in 100% of ICCs, 52.6% of ECCs, and in 38.5% of GBCs. P-MAPK and p-Akt are highly expressed in ICCs (>58% of samples), and to a lower extent in ECCs and GBCs (<46%), indicating EGFR pathway activation. HER2 is overexpressed in 10% of GBCs (with genomic amplification), and 26.3% of ECCs (half of which has genomic amplification). EGFR or its signal transducers are mutated in 26.5% of cases: 4 samples bear mutations of PI3K (8.2%), 3 cases (6.1%) in K-RAS, 4 (8.2%) in B-RAF, and 2 cases (4.1%) in PTEN, but no loss of PTEN expression is detected. EGI-1 cell line is highly sensitive to gemcitabine, TFK1 and TGBC1-TKB cell lines are responsive and HuH28 cell line is resistant. In EGI-1 cells, combination with gefitinib further increases the antiproliferative effect of gemcitabine. In TFK1 and TGBC1-TKB cells, the efficacy of gemcitabine is increased with addiction of sorafenib and everolimus. In TGBC1-TKB cells, lapatinib also has a synergic effect with gemcitabine. HuH28 becomes responsive if treated in combination with erlotinib. Moreover, HuH28 cells are sensitive to lapatinib as a single agent. Molecular mechanisms were confirmed by western blot analysis.
  
  
  CONCLUSIONS
  These data demonstrate that EGFR and HER2 pathways are suitable therapeutic targets for BTCs. The combination of gemcitabine with drugs targeting these pathways gives encouraging results and further clinical studies could be warranted.
authors.affiliation: Department of Medical Oncology, University of Torino Medical School, Institute for Cancer Research and Treatment, Candiolo, Italy. ymera.pignochino@ircc.it
authors.forename: Ymera
authors.initials: Y
authors.lastname: Pignochino
authors.forename: Ivana
authors.initials: I
authors.lastname: Sarotto
authors.forename: Caterina
authors.initials: C
authors.lastname: Peraldo-Neia
authors.forename: Junia Y
authors.initials: JY
authors.lastname: Penachioni
authors.forename: Giuliana
authors.initials: G
authors.lastname: Cavalloni
authors.forename: Giorgia
authors.initials: G
authors.lastname: Migliardi
authors.forename: Laura
authors.initials: L
authors.lastname: Casorzo
authors.forename: Giovanna
authors.initials: G
authors.lastname: Chiorino
authors.forename: Mauro
authors.initials: M
authors.lastname: Risio
authors.forename: Alberto
authors.initials: A
authors.lastname: Bardelli
authors.forename: Massimo
authors.initials: M
authors.lastname: Aglietta
authors.forename: Francesco
authors.initials: F
authors.lastname: Leone
mesh_terms: D000328:Adult; D000368:Aged; D000369:Aged, 80 and over; D000964:Antimetabolites, Antineoplastic / Q000494:pharmacology; D000971:Antineoplastic Combined Chemotherapy Protocols / Q000494:pharmacology*; D001661:Biliary Tract Neoplasms / Q000201:enzymology* / Q000235:genetics / Q000473:pathology; D015153:Blotting, Western; D002277:Carcinoma / Q000201:enzymology* / Q000235:genetics / Q000473:pathology; D045744:Cell Line, Tumor; D049109:Cell Proliferation / Q000187:drug effects*; D003841:Deoxycytidine / Q000031:analogs & derivatives / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D066246:ErbB Receptors / Q000037:antagonists & inhibitors* / Q000378:metabolism; D005260:Female; D005706:Gallbladder Neoplasms / Q000201:enzymology* / Q000235:genetics / Q000473:pathology; D005784:Gene Amplification; D015972:Gene Expression Regulation, Neoplastic; D006801:Humans; D007150:Immunohistochemistry; D017404:In Situ Hybridization, Fluorescence; D047908:Intracellular Signaling Peptides and Proteins / Q000235:genetics / Q000378:metabolism; D008297:Male; D008875:Middle Aged; D058990:Molecular Targeted Therapy; D009154:Mutation; D047428:Protein Kinase Inhibitors / Q000494:pharmacology; D018719:Erb-b2 Receptor Tyrosine Kinases / Q000037:antagonists & inhibitors* / Q000378:metabolism; D015398:Signal Transduction / Q000187:drug effects* / Q000235:genetics; D000093542:Gemcitabine
chemical_list: D000964:Antimetabolites, Antineoplastic; D003841:Deoxycytidine; D066246:ErbB Receptors; D047908:Intracellular Signaling Peptides and Proteins; D047428:Protein Kinase Inhibitors; D018719:Erb-b2 Receptor Tyrosine Kinases; D000093542:Gemcitabine; C512478:EGFR protein, human; C508053:ERBB2 protein, human