Primary tissue biopsies are often prepared as formalin-fixed paraffin-embedded (FFPE) samples. FFPE preps have the advantage of being amenable to indefinite storage at room temperature allowing the nucleic acids (both DNA and RNA) to be recovered and analyzed, even decades later. However, FFPE samples have been notoriously difficult to work with because formalin fixation can cause nucleic acid degradation, or damage, and often there is too little tumor DNA/RNA in the sample. Improvements in molecular analytical methods, such as real-time PCR, arrays, and even next generation sequencing (NGS) have resulted in better sensitivity but these analytical methods are still only as good as the quality and quantity of the nucleic acid sample used.
Characterizing Primary Tumor Mutations
Accurate assessment of the presence and frequency of any given mutation is dependent on many factors including:
the heterogeneity within the tumor for the target mutation, i.e. the prevalence of the mutation amongst the tumor cells;
the number of tumor cells relative to all the other cells sampled
DNA Extraction Is Non-Specific
The most commonly used method for molecular analysis of FFPE samples relies on DNA extraction from slices of an FFPE tissue sample. The FFPE tissue slice is typically “de-paraffinized” and the DNA is extracted from all the cells on the slice. The result is a characterization or analysis of all DNA present, including DNA from tumor cells, stromal cells, infiltrating lymphocytes, etc. The frequency of a given mutation is an estimate at best.
Separate Tumor Cells from Stromal Cells
To achieve a more accurate assessment of the prevalence of various mutations, the DEPArray system can be used to separate tumor cells from stromal, and other, cells in the FFPE section. A slice or punch from an FFPE sample can be de-paraffinized and the suspension of cells stained for various cell types, e.g. keratin and vimentin. Using the DEPArray system’s image-based sorting capabilities, tumor cells can be separated from the stromal cells that were present in the tissue section. A highly pure collection of tumor cells, e.g. 300 cells, can be recovered from FFPE tissue samples to achieve the most accurate and sensitive molecular characterization. By recovering fully intact cells, there is sufficient DNA in 300 cells to go directly to next generation sequencing (NGS) of a cancer hotspot panel.
FFPE Application Workflow
The complete FFPE application workflow includes a sample preparation kit and a DNA quality control assay for FFPE samples - DEPArray™ FFPE SamplePrep Kit and DEPArray™ FFPE QC Kit - a cell lysis kit downstream of DEPArray™ sorting - DEPArray™ LysePrep Kit - and an oncology panel library preparation kit for Next Generation Sequencing (NGS) – DEPArray™ OncoSeek Panel.
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