Exploring allelic imbalance within paraffin-embedded tumor biopsies using pyrosequencing technology.
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).
Pascual
Manuel Hidalgo
MH
Servicio de Cirugía General B, Hospital Universitario 12 de Octubre, Madrid, Spain.
Royo
José Luis
JL
Martínez-Tello
Francisco Jose
FJ
Crespo
Carmen
C
Salinas
Ana
A
Herrero
Eduardo Ferrero
EF
Lopez-García
María
M
Real
Luis Miguel
LM
Ruiz
Agustín
A
Ramirez-Lorca
Reposo
R
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
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