title: Tissue microarrays: applications in study of p16 and p53 alterations in Ewing's cell lines.
abstract: BACKGROUND
  Tissue microarrays (TMAs) are used to study genomics and proteomics in several tumour tissue samples. Cell lines (CC) are of great importance in the study of the genetic changes in tumours, and some reveal several aspects of tumour oncogenesis. There are few published reports on Ewing's tumours with TMAs including original tumours (OT) and corresponding CC.
  
  
  METHODS
  We have performed four TMAs, from 3 OT and the corresponding CC of successive in vivo and in vitro tumour passages. Xenotransplant CC in nude mice from OT (XT/OT) was made. Subsequently multiple XT were performed and in vitro XT cell line (CC/XT) was obtained. In vivo re-inoculation of CC/XT (XT/CC) was planned. TMAs with the successive tumour passages that grew in nude mice (XT/OT and XT/CC) were analyzed by morphologic pattern (Hematoxilin/eosin), immunohistochemical staining (CD99, FLI1, p16, p53, ki-67), fluorescent in situ hybridization-FISH-(EWSR1 break apart, p16 and p53 status) and gene fusion types.
  
  
  RESULTS
  Heterogeneous results of the p16, p53 and ki67 in OT, XT/OT, CC/XT and XT/CC were observed. The three cell lines revealed EWS/FLI1 rearrangements. p16 gene was deleted only in one case. The deletion was detected by FISH and confirmed by PCR assays. A p53 alteration was found in the second case with monosomy and subsequently polysomic status of chromosome 17 during the evolution of CC. The PCR study revealed p53 mutation. The third case showed hypermethylation in the promoter of p16. The growth of the tumour in nude mice was more accelerated when the inoculation was performed from the CC/XT, increasing progressively over the passages. The third case did not reveal tumour growth in nude mice after the re-inoculation of CC/XT.
  
  
  CONCLUSIONS
  The study of several cores from original tumours and successive tumour passages in TMAs facilitated the analysis of the genetic alteration and protein expression in Ewing's tumours.
authors.affiliation: Department of Pathology, University of Valencia, Valencia, Spain. rosa.noguera@uv.es
authors.forename: Rosa
authors.initials: R
authors.lastname: Noguera
authors.forename: Isidro
authors.initials: I
authors.lastname: Machado
authors.forename: Marta
authors.initials: M
authors.lastname: Piqueras
authors.forename: Jose Antonio
authors.initials: JA
authors.lastname: Lopez-Guerrero
authors.forename: Samuel
authors.initials: S
authors.lastname: Navarro
authors.forename: Empar
authors.initials: E
authors.lastname: Mayordomo
authors.forename: Antonio
authors.initials: A
authors.lastname: Pellin
authors.forename: Antonio
authors.initials: A
authors.lastname: Llombart-Bosch