Abstract B51: Extraction and analysis of genomic DNA from formalin-fixed paraffin-embedded neuroblastoma samples following laser capture microdissection
Authors: Thwe Le M. , Cantrill Laurence C. , Catchpoole Daniel R. , Lau Loretta , Byrne Jennifer A. 
Published: May 2015

Abstract
Introduction: Neuroblastoma (NB) is the most common extracranial solid tumor of infants. We constructed a tissue microarray (TMA) including 47 diagnostic NB cases and performed immunohistochemical (IHC) staining for a number of biomarkers. Differential IHC staining was identified between duplicate tissue cores for a proportion of NB cases. Differential IHC staining occurs frequently in cancer specimens, and can be due to technical factors, tissue artefacts, or underlying biological differences between tumor sub-regions. As laser capture microdissection (LCM) allows isolation of cells from formalin-fixed paraffin-embedded (FFPE) tissue slides, we applied LCM to FFPE NB samples, to investigate the basis of differential IHC staining.
Material and Methods: NSE, NB84, ALK, TPD52 and MGMT proteins were detected within diagnostic NB TMA cores and whole NB sections using IHC. Cells were isolated from FFPE sections using LCM, and genomic DNA was extracted using the QIAamp DNA FFPE Tissue Kit. Extracted genomic DNA was quantified using a Nanodrop spectrophotometer, and DNA quality was assessed by a Bioanalyzer 2100. All DNA samples were tested using PCR with TPD52 and ALK primers. Results including clinical and sample variables were analysed using SPSS version 19.
Results: Of 47 NB cases within the TMA, 35 cases were represented by duplicate tissue cores, whereas the remaining 12 cases were represented by single cores. IHC analysis of 5 biomarkers showed visually different IHC staining in tumour tissue between duplicate tissue cores in 15/35 (43%) cases. Whole tissue sections from 6 of these 15 cases were then examined, which reproduced differential IHC staining. Isolation of NB cells by LCM was successfully performed after substantial optimisation. A total of 14 regions from 8 NB cases were microdissected, producing DNA yields ranging from 0.12 - 4.05 pg/ µm2 tissue. There were no significant associations between total yields of extracted DNA, and microdissected tissue areas, or tissue sample ages. All DNA samples were successfully amplified by TPD52 but not by ALK PCR primers.
Conclusions: Genomic DNA can be reliably extracted from microdissected FFPE NB tissues, although only one of 2 genes could be successfully PCR amplified. Downstream assays of DNA extracted from FFPE samples may therefore have particular design requirements. Further application of this approach may help improve our understanding of the level of tumour heterogeneity present in NB samples at diagnosis.
Citation Format: Le M. Thwe, Laurence C. Cantrill, Daniel R. Catchpoole, Loretta Lau, Jennifer A. Byrne. Extraction and analysis of genomic DNA from formalin-fixed paraffin-embedded neuroblastoma samples following laser capture microdissection. [abstract]. In: Proceedings of the AACR Special Conference on Pediatric Cancer at the Crossroads: Translating Discovery into Improved Outcomes; Nov 3-6, 2013; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2013;74(20 Suppl):Abstract nr B51.