title: Expression of cellular FLICE inhibitory protein, caspase-8, and protease inhibitor-9 in Ewing sarcoma and implications for susceptibility to cytotoxic pathways.
abstract: OBJECTIVE
  Ewing sarcoma is a common pediatric bone tumor with an unfavorable prognosis for metastatic or recurrent disease. Cellular immunotherapy may provide new treatment options and depends on the cytolytic death receptor and perforin/granzyme pathways. Expression of death receptor pathway inhibitor cellular FLICE inhibitory protein (cFLIP), initiator caspase-8, and granzyme B inhibitor protease inhibitor-9 (PI-9) have been reported to determine susceptibility to cell- and chemotherapy-mediated killing in several tumor types. Here, we have studied their in vitro and in vivo expression in Ewing sarcoma and the implications for susceptibility to cytotoxicity.
  
  
  METHODS
  Ewing sarcoma cell lines (n = 8) were tested for cFLIP, PI-9, and caspase-8 expression. Functional significance was tested by anti-Fas antibody (death receptor pathway) or natural killer cell (perforin/granzyme pathway) treatment. Immunohistochemistry was done on 28 sections from 18 patients. In half of the cases, sequential material, including metastases, was available.
  
  
  RESULTS
  Although all tested Ewing sarcoma cell lines expressed cFLIP, resistance to CD95/Fas-mediated apoptosis was only observed in two cell lines lacking caspase-8 expression. PI-9 was expressed at low levels in four of eight Ewing sarcoma cell lines, but positive cell lines remained susceptible to perforin/granzyme-mediated killing. In primary Ewing sarcoma, including metastases, cFLIP was abundantly expressed in 18 of 18 patients. Caspase-8 was expressed in all patients but showed more intertumoral and intratumoral variation in both intensity and heterogeneity of staining. PI-9, in contrast, was undetectable.
  
  
  CONCLUSIONS
  The expression patterns of cFLIP, caspase-8, and the absence of PI-9 provide a rationale to preferentially exploit the perforin/granzyme pathway in cytotoxic therapies against Ewing sarcoma.
authors.affiliation: Department of Pediatrics, Division of Immunology, Haematology, Oncology, Bone Marrow Transplantation and Autoimmune Diseases, Leiden University Medical Center, Leiden, the Netherlands.
authors.forename: Alfons S K
authors.initials: AS
authors.lastname: de Hooge
authors.forename: Dagmar
authors.initials: D
authors.lastname: Berghuis
authors.forename: Susy Justo
authors.initials: SJ
authors.lastname: Santos
authors.forename: Esther
authors.initials: E
authors.lastname: Mooiman
authors.forename: Salvatore
authors.initials: S
authors.lastname: Romeo
authors.forename: J Alain
authors.initials: JA
authors.lastname: Kummer
authors.forename: R Maarten
authors.initials: RM
authors.lastname: Egeler
authors.forename: Maarten J D
authors.initials: MJ
authors.lastname: van Tol
authors.forename: Cornelis J M
authors.initials: CJ
authors.lastname: Melief
authors.forename: Pancras C W
authors.initials: PC
authors.lastname: Hogendoorn
authors.forename: Arjan C
authors.initials: AC
authors.lastname: Lankester
mesh_terms: D017209:Apoptosis; D014408:Biomarkers, Tumor; D001706:Biopsy; D053446:CASP8 and FADD-Like Apoptosis Regulating Protein / Q000096:biosynthesis*; D053181:Caspase 8 / Q000096:biosynthesis* / Q000378:metabolism; D045744:Cell Line, Tumor; D053938:DNA Fragmentation; D015972:Gene Expression Regulation, Neoplastic; D020022:Genetic Predisposition to Disease; D006801:Humans; D007150:Immunohistochemistry; D007167:Immunotherapy / Q000379:methods; D012512:Sarcoma, Ewing / Q000235:genetics* / Q000378:metabolism*; D015843:Serpins / Q000096:biosynthesis*; D019014:fas Receptor / Q000096:biosynthesis
chemical_list: D014408:Biomarkers, Tumor; D053446:CASP8 and FADD-Like Apoptosis Regulating Protein; C102316:SERPINB9 protein, human; D015843:Serpins; D019014:fas Receptor; D053181:Caspase 8