(SW4) Use of FFPE in Next-Generation Sequencing: Considerations from Bedside to Bench
FFPE Sequencing and Bioinformatics
Next generation sequencing has identified a number of clinically relevant alterations in cancer predominantly using fresh frozen (FF) specimens. Extending this capability to FFPE specimens requires a thorough understanding of the consequence of formalin-fixation on the analysis of genomic data. In TCGA, we compared multi-platform molecular signatures of FFPE preservation to paired FF specimens as the ‘gold standard’. DNA and RNA were obtained from six different cancer types across 38 patients using a co-isolation method optimized for FFPE. These samples were assessed for effects of formalin-fixation on whole exome sequencing, DNA copy number, DNA methylation, mRNA and miRNA sequencing. Considerations for analysis and usability of data were determined for each data type.
Whole Exome Sequencing analysis of Archival Formalin Fixed Paraffin Embedded Tissue (FFPE): comparison of different library preparation methods
Formalin Fixed Paraffin Embedded Tissue (FFPE) procurement is the standard for tumor banking and remains part of clinical standard of care. These samples are an invaluable resource which provides an excellent opportunity to progress cancer research with well characterized histological and pathological annotation combined with extensive clinical data. Exploitation of FFPE samples utilizing Next Generation Sequencing approaches has been challenging. Great strides using targeted DNA sequencing strategies to screen known actionable mutations in FFPE samples have been accomplished however the use of DNA extracted from FFPE for whole exome sequencing (WES) is presently limited. The ability to use archival FFPE samples to screen entire exomes for both known and novel mutations will have strong impact on clinical and basic research initiatives. The pre-capture PCR step within the WES protocol is the most critical when working with degraded samples, which can therefore significantly affect the quality of sequencing data and downstream analysis. To increase efficacy of template production we have tested several different methods to add adaptors (pre-capture PCR) within the WES protocol.
Active organic solvent-free paraffin removal is the key to standardizing extraction of biomolecules from FFPE tissues
Formalin Fixation and Paraffin Embedding (FFPE) of tissues, a mainstay of clinical histological analysis for the past century, has been rapidly adopted for targeted and whole genome sequencing of clinical oncology samples for tumor mutation identification, targeted therapy choice, and treatment monitoring. The use of extreme formaldehyde fixation, not envisioned for use in molecular diagnostics, presents not only a technical challenge to reproducible DNA,RNA, and protein extraction for molecular analysis, but also a workflow challenge for protocol standardization in a clinical setting due to the intensive and manual steps required for passive removal of the paraffin embedding medium.
In this talk I will present a highly reproducible, rapid, efficient, and easily automatable method of extraction and purification of biomolecules from FFPE tissue utilizing Covaris Adaptive Focused Acoustics (AFA). This unique methodology utilizes highly controlled acoustic energy for active and effective removal of paraffin from FFPE cores, sections, and slides enabling efficient and rapid tissue rehydration, tissue digestion, crosslink reversal, and biomolecule release. This critical step is carried out rapidly without the use of dangerous organic solvents or messy mineral oils, significantly improving the FFPE sample processing workflow, and greatly reducing the possibility of cross contamination and handling errors.
I will also share internal Covaris data, as well as peer-reviewed publication data illustrating the significant effect of active paraffin removal on downstream applications and analyses of DNA, RNA and proteins from FFPE tissues. The simplified single-tube method and rapid workflow allows for the parallel processing of 8 to 96 FFPE samples in a batch format easily adaptable to the throughput requirement of medium to high volume clinical labs and core facilities.