Immuno-Guided Laser Assisted Microdissection Techniques for Formalin-Fixed Tissue Samples
FC Eberle, JC Hanson, JK Killian, K Ylaya, SM Hewitt, ES Jaffe, MR Emmert-Buck, J Rodriguez-Canales. National Cancer Institute, Bethesda, MD
Background: Laser assisted microdissection (LAM) has been incorporated in pathology as a tool for molecular profiling. Immuno-guided LAM (I-LAM) is a technique for isolating specific cells after immunohistochemistry (IHC). Current LAM systems incorporate a UV-based cutting laser that requires special membrane slides. For formalin-fixed paraffin embedded (FFPE) specimens the use of membrane slides with standard IHC techniques is not suitable. Moreover, the effects of IHC on both DNA integrity and methylation profile for epigenetic analysis of FFPE specimens are unknown. The goal of this study is to establish an IHC protocol that is compatible with I-LAM of FFPE specimens, and to evaluate the effects of this technique on DNA integrity and methylation profile.
Design: By using 10 FFPE samples (5 primary mediastinal large B-cell lymphoma and 5 classical Hodgkin's lymphoma) we tested different methods of slide preparation, antigen retrieval (AR) and IHC. Tissue sections were either stained with H&E (for LAM) or with anti-CD20 or anti-CD30 (for I-LAM) before tumor cells were microdissected using Leica LMD 6000. DNA was extracted, evaluated for amount and integrity and the global DNA methylation profile of H&E or IHC stained tumor cells of each individual sample was compared using a CpG array platform (Illumina GoldenGate Methylation Array).
Results: The best technique for membrane slide preparation was achieved by UV-irradiation and poly-L-lysine coating. Testing different AR buffers, distinct incubation times and antibody dilutions, we could demonstrate specific target cell staining with high reproducibility in all 10 cases. After following the optimized protocol, tumor cells were isolated by LAM or I-LAM. PCR of extracted DNA from tumor cells showed that DNA integrity was not affected by AR and IHC compared to H&E controls. Global epigenetic profile was not altered by IHC in comparison with H&E showing strong correlation (r = 0.945 to 0.981) for CpG target methylation of 1505 analyzed sites.
Conclusions: These results demonstrate the validity and utility of our IHC protocol for I-LAM. IHC does not affect DNA integrity and the epigenetic profile compared to H&E staining. This protocol allows the application of I-LAM for specific genomic and epigenetic analysis from FFPE specimens.
Monday, March 22, 2010 1:00 PM
Poster Session II # 258, Monday Afternoon