Deep oncopanel sequencing reveals within block position-dependent quality degradation in FFPE processed samples

Area of Science:

• Oncology
• Molecular Diagnostics
• Biotechnology

Background:

• Clinical laboratories utilize formalin-fixed paraffin-embedded (FFPE) samples for oncology panel sequencing.
• FFPE processing is crucial for identifying mutations that guide targeted therapy selection.
• Understanding technical errors introduced by FFPE processing is vital for accurate genomic profiling.

Purpose of the Study:

• To investigate the technical errors associated with formalin fixation and paraffin embedding (FFPE) in oncology panel sequencing.
• To evaluate the impact of varying formalin fixation times on sample integrity.
• To identify sources of technical variability in FFPE sample analysis.

Main Methods:

• A characterized diploid cell line was used to generate FFPE samples with diverse fixation times.
• Ninety-six FFPE sections were distributed to multiple laboratories for targeted sequencing.
• Four distinct oncopanels were employed for variant analysis to detect technical errors.

Main Results:

• Tissue sections with low cellularity, insufficient DNA input, or inadequate sequencing depth were more prone to errors.
• FFPE samples from block surfaces exhibited higher rates of FFPE-specific technical errors, similar to histological "edge effects".
• Inner FFPE samples showed no quality degradation linked to fixation duration.

Conclusions:

• Avoiding the use of FFPE block surface portions is recommended for reliable sequencing results.
• Restricting mutation detection to high-confidence genomic regions can enhance accuracy.
• Implementing these strategies will improve the reliability of targeted therapy selection based on FFPE sample analysis.