Abstract 820: Genome-wide aberration analysis of rare cancer cell populations derived from formalin fixed paraffin embedded tissue.

Introduction. The majority of clinically described patient material worldwide is available as formalin fixed parafin embedded (FFPE) tissue blocks. Despite being a cheap and convenient way of archiving tissue suitable for review under the microscope, FFPE blocks are difficult to interrogate with modern molecular biology methods due to changes occurring during the process of fixation, embedding and aging. Genetic analysis of tumours with limited malignant cell count, like classical Hodgkin lymphoma (cHL) or T-cell/histiocyte-rich B cell lymphoma (TCHRBCL) is impeded by a bulk mass of non-malignant tumour infiltrating cells. Laser capture microdissection is currently a method of choise for rare tumor cell isolation, however it induce serious damage for gDNA, therefore additional techniques for enrichment are needed to enable meaningful genome-wide aberration analysis of tumors with low cancer cell content. Methods. For extraction of nuclei from FFPE blocks of cHL and TCHRBCL cases enzymatic digestion and mechanical force were utilized. Pure populations of cancer cell nuclei were isolated by fluorescence-activated cell sorting (FACS) based on cancer type-specific nuclear markers Pax-5, Mum-1, Bcl-6, Ki-67. Array comparative genomic hybridization (aCGH) was utilized for genome-wide aberration analysis. Results. We have optimized a set of methods to successfully isolate intact, high quality nuclei from FFPE tissue blocks. Nuclei retain their antigenicity and, therefore, are suitable for labelling with frourophore-conjugarted antibodies, which enables flow-sorting based not only on physical parameters and DNA content, but also on positivity for highly specific nuclear markers. Tumour nuclei populations from cHL and TCHRBCL of 70-90% in purity were isolated. DNA extracted from as little as 500 flow-sorted nuclei was sufficient to generate high-resolution and reproducible aCGH data. Conclusions. Our proposed methodology allows the use of tissues in FFPE blocks more widely. Efficient isolation of malignant cells from the tumour mass was achieved. In the cases where starting material is scarce and very low number of target nuclei can be isolated, DNA extraction with improved recovery together with commercially available whole genome amplification methods enable genome-wide aberration analysis. Citation Format: Darius Juskevicius, Christian Ruiz, Tanja Dietsche, Alex Rufle, Michael T. Barrett, Lukas Bubendorf, Stephan Dirnhofer, Alexander Tzankov. Genome-wide aberration analysis of rare cancer cell populations derived from formalin fixed paraffin embedded tissue. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 820. doi:10.1158/1538-7445.AM2013-820