title: Exploring allelic imbalance within paraffin-embedded tumor biopsies using pyrosequencing technology.
abstract: BACKGROUND
  The comparison of molecular genetic changes in healthy and pathological tissues has historically led to the identification of oncogenes and tumor suppressor genes. It is very common that studies investigating loss of heterozygosity are carried out retrospectively on paraffin-embedded samples.
  
  
  METHODS
  In this study, we evaluated the power of pyrosequencing for determining the loss of heterozygotic regions. The present method uses the fact that pyrosequencing is an accurate, sensitive and reproducible technique. The method is also simple to perform, with results available in 96-well format, making the assays amenable to automation. Thus, we analyzed nine single nucleotide polymorphisms along 1 Mb between the EMSY and PAK1 genes on 11q13, a region frequently rearranged in different tumors and cell lines. We assessed the study using samples from breast cancer and thyroid cancer biopsies.
  
  
  CONCLUSIONS
  We conclude that this technique is capable of detecting variations of >10% in allele loss. However, strong allele imbalances were detected, depending on the origin of the sample. Seven out of the nine markers used exhibited differential allele amplification, depending on the DNA quality (p<0.01).
authors.affiliation: Servicio de Cirugía General B, Hospital Universitario 12 de Octubre, Madrid, Spain.
authors.forename: Manuel Hidalgo
authors.initials: MH
authors.lastname: Pascual
authors.forename: José Luis
authors.initials: JL
authors.lastname: Royo
authors.forename: Francisco Jose
authors.initials: FJ
authors.lastname: Martínez-Tello
authors.forename: Carmen
authors.initials: C
authors.lastname: Crespo
authors.forename: Ana
authors.initials: A
authors.lastname: Salinas
authors.forename: Eduardo Ferrero
authors.initials: EF
authors.lastname: Herrero
authors.forename: María
authors.initials: M
authors.lastname: Lopez-García
authors.forename: Luis Miguel
authors.initials: LM
authors.lastname: Real
authors.forename: Agustín
authors.initials: A
authors.lastname: Ruiz
authors.forename: Reposo
authors.initials: R
authors.lastname: Ramirez-Lorca
mesh_terms: D022981:Allelic Imbalance / Q000235:genetics*; D001331:Automation; D001483:Base Sequence; D001706:Biopsy / Q000379:methods; D002874:Chromosome Mapping; D002880:Chromosomes, Human, Pair 11 / Q000235:genetics; D006579:Heterozygote; D006801:Humans; D009363:Neoplasm Proteins / Q000235:genetics; D009369:Neoplasms / Q000235:genetics / Q000473:pathology*; D009687:Nuclear Proteins / Q000235:genetics; D016612:Paraffin Embedding / Q000379:methods; D020641:Polymorphism, Single Nucleotide; D017346:Protein Serine-Threonine Kinases / Q000235:genetics; D012097:Repressor Proteins / Q000235:genetics; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D017422:Sequence Analysis, DNA / Q000379:methods*; D054462:p21-Activated Kinases
chemical_list: C480344:EMSY protein, human; D009363:Neoplasm Proteins; D009687:Nuclear Proteins; D012097:Repressor Proteins; C515903:PAK1 protein, human; D017346:Protein Serine-Threonine Kinases; D054462:p21-Activated Kinases