Immunoguided laser assisted microdissection techniques for DNA methylation analysis of archival tissue specimens.
Altered DNA methylation is a fundamental characteristic of carcinogenesis. The analysis of DNA methylation in tumor cells may help to better understand tumor pathogenesis and more importantly may be used as diagnostic tool with therapeutic consequences. To detect targets relevant in tumorigenesis, it is essential to separate neoplastic cells from nonneoplastic cells. An excellent method for isolating specific cells is laser-assisted microdissection (LAM). Target cell identification for immunoguided LAM (ILAM) requires immunohistochemistry (IHC). Yet, it is unclear whether IHC for ILAM influences DNA methylation. The goals of this study were to establish an optimized protocol for antigen retrieval and IHC of formalin-fixed paraffin-embedded (FFPE) specimens suitable for ILAM and to evaluate its effect on the DNA methylome using a high throughput array. Using ten archival FFPE specimens, we showed specific staining suitable for ILAM. Extracted DNA from microdissected cells of immunohistochemically or H&E-stained tissue sections showed identical DNA quality and a strong correlation (r = 0.94 to 0.98) for CpG target methylation of 1505 analyzed sites in a series of five paired samples. No differential methylation between H&E and IHC was detected in 1501 of 1505 CpG targets (99.7%; P < 0.05). These results demonstrate the validity and utility of the herein described protocol, which allows the application of ILAM for large-scale genomic and epigenetic analyses of archival tissue specimens.
Eberle
Franziska C
FC
Hematopathology Section, National Cancer Institute, National Institutes of Health, 8717 Grovemont Circle, Bethesda, MD 20892, USA.
Hanson
Jeffrey C
JC
Killian
J Keith
JK
Wei
Lai
L
Ylaya
Kris
K
Hewitt
Stephen M
SM
Jaffe
Elaine S
ES
Emmert-Buck
Michael R
MR
Rodriguez-Canales
Jaime
J
D000951:Antigens, Neoplasm / Q000276:immunology; D018070:Biological Specimen Banks; D018899:CpG Islands / Q000235:genetics; D019175:DNA Methylation / Q000235:genetics*; D004273:DNA, Neoplasm / Q000235:genetics; D006689:Hodgkin Disease / Q000235:genetics / Q000276:immunology / Q000473:pathology; D006801:Humans; D007150:Immunohistochemistry; D017730:Ki-1 Antigen / Q000378:metabolism; D007834:Lasers; D042282:Microdissection / Q000379:methods*; D016133:Polymerase Chain Reaction; D013194:Staining and Labeling
D000951:Antigens, Neoplasm; D004273:DNA, Neoplasm; D017730:Ki-1 Antigen