Formalin Fixation, Delay to Fixation, and Time in Fixative Adversely Impact Copy Number Variation Analysis by aCGH

Area of Science:

• Oncology
• Genetics
• Molecular Biology

Background:

• Molecular profiling of DNA from formalin-fixed, paraffin-embedded (FFPE) tumor specimens is increasingly utilized.
• The impact of FFPE processing variables on copy number variation (CNV) detection remains incompletely understood.

Purpose of the Study:

• To investigate how different delays to fixation (DTF) and times in fixative (TIF) affect CNV detection in FFPE renal cell carcinoma specimens.
• To compare CNV profiles from FFPE specimens with those from matched snap-frozen tumor and blood samples.

Main Methods:

• Array comparative genomic hybridization (aCGH) was performed on FFPE and snap-frozen tumor specimens.
• FFPE specimens were processed with varying DTFs (1-12 hours) and TIFs (6-72 hours).
• CNV profiles were analyzed and compared between FFPE and frozen samples, considering DNA characteristics like GC content.

Main Results:

• FFPE specimens revealed more CNV regions compared to matched snap-frozen specimens.
• Extended DTF (≥3 hours) increased the number of segments with differing copy number status, while prolonged TIF (72 hours) decreased it.
• Stable CNV regions in FFPE samples showed higher guanine-cytosine (GC) content and/or repeat percentages.

Conclusions:

• FFPE processing variables significantly influence CNV detection, with DTF and TIF being critical factors.
• To mitigate these effects, FFPE analysis should ideally use specimens with DTF <3 hours and TIF <72 hours.
• Validation of FFPE-derived results against fresh or frozen specimens is recommended whenever feasible.