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.Epigenetics is defined as the study of heritable changes in gene expression that are not attributable to alterations in the DNA sequence.¹ The best-known epigenetic marker is DNA methylation. DNA methylation plays an important role in the control of gene activity and the architecture of the nucleus.² During carcinogenesis, DNA of tumor cells is characterized by hypomethylation in gene regions that promote tumor development, whereas hypermethylation occurs among tumor suppressor genes.^3,4 Especially for large-scale analysis of cancer tissue methylation, it is important to investigate the tumor cells and not the intermixed inflammatory cells or other normal host cells to identify targets relevant in tumorigenesis. Therefore, laser-assisted microdissection (LAM) is a helpful tool to isolate cell populations for cell-specific molecular profiling.⁵ Typically, the identification of target cells relies on morphological cell characteristics observed after routine histological staining. The gold standard for histopathological diagnosis is hematoxylin-eosin (H&E) staining.⁶ However, depending on the type of tissue and the cells of interest, H&E staining may be inadequate for a clear identification of some cell types. Optical resolution is especially impaired when using the LAM scope, because LAM is not compatible with use of a cover slide and selection of cells with less distinct morphological characteristics can be hampered under these conditions. In cases where routine staining does not allow discrimination between normal and pathological cell morphology, visualization of cell surface or intracellular structures can be helpful. For example, labeling of cellular proteins by immunohistochemistry (IHC) can assist in identification of specific target cells. The application of IHC to LAM, also called immunoguided LAM (ILAM), is a valuable technique that allows localization of target cells and microdissection of particular cell populations. In addition, IHC enables the use of automatic cell recognition programs included in the advanced series of LAM instruments. By using LAM devices with automatic cell recognition programs it is technically feasible to collect high numbers of target cells in a reasonable time.⁷Currently antigen retrieval (AR) and IHC on positively charged glass slides is a widely established method in routine pathology laboratories. The original laser capture microdissection system was based on an infrared (IR) laser that captured the cells of interest from tissue sections mounted on glass slides.⁵ However, all current microdissection systems incorporate an UV cutting laser that is not suitable for positively charged glass slides but requires special polyethylene naphthalate (PEN) membrane slides. IHC on membrane slides, particularly common AR procedures required for formalin-fixed paraffin embedded (FFPE) tissue samples, usually result in loss of parts of the tissue or the entire tissue section. For that reason regularly used AR methods at high temperature (90°C) are not suitable for ILAM. More importantly, it is unclear whether IHC for ILAM influences DNA integrity or DNA methylation of target cells from archival tissue samples. The goals of this study were to establish an optimized protocol for AR and IHC of FFPE tissue samples suitable for ILAM and to determine whether the DNA methylation profile is affected by AR or IHC. For establishing this protocol, we selected FFPE specimens from primary mediastinal large B-cell lymphoma (PMLBCL) as well as cases of classical Hodgkin''s lymphoma (CHL), the latter representing a model of a highly challenging LAM.