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Use of formalin-fixed paraffin-embedded tumor tissue as a DNA source in molecular epidemiological studies of
Anthea Elizabeth Ferguson1, Richard Julian Cohn, Lesley Jayne Ashton
1Molecular Epidemiology Group, Children's Cancer Institute Australia, Randwick, NSW, Australia.
Abstract:
Formalin-fixed paraffin-embedded tissue (FFPET) samples are a potential source of DNA for molecular epidemiological studies. However, the use of FFPET samples can be restricted by the yield and quality of DNA isolated. The aim of this study was to examine whether FFPET biopsies from pediatric central nervous system tumors were a feasible alternative to archival frozen tissue when characterizing common gene polymorphisms. DNA was isolated from 50 frozen pediatric central nervous system tumor biopsies and matched FFPET samples. Real-time polymerase chain reaction (PCR) was used to quantify DNA and characterize GSTT1, GSTM1, GSTP1, and MTHFR gene polymorphisms. The use of whole-genome amplification (WGA) to increase DNA yields was also investigated. The results showed that DNA isolated from FFPET samples was more fragmented and provided smaller yields than DNA isolated from frozen samples. Attempts to increase the DNA yield from FFPET using WGA were unsuccessful. DNA from FFPET samples was successfully genotyped for the GSTP1 Ile105Val and MTHFR 677 C>T polymorphisms in 98% of samples and was 100% concordant with the results from frozen tissue. However, DNA from FFPET performed poorly in real-time PCR assays for GSTM1 and GSTT1 deletion polymorphisms. Our investigations show that DNA extracted from FFPET is substantially fragmented and not readily amplified using WGA. In addition, careful validation of PCR assays should be carried out due to the variable amplification of fragmented FFPET DNA.
Insights
Formalin-fixed paraffin-embedded tissue (FFPET) DNA can be used for gene polymorphism studies in pediatric brain tumors. However, FFPET DNA quality and yield are variable, impacting PCR assay success.
Area of Science:
- Molecular Epidemiology
- Genetics
- Oncology
Background:
- Formalin-fixed paraffin-embedded tissue (FFPET) is a valuable resource for DNA extraction in molecular epidemiological studies.
- Challenges exist in obtaining sufficient DNA yield and quality from FFPET samples for genetic analysis.
Purpose of the Study:
- To evaluate the feasibility of using FFPET biopsies from pediatric central nervous system tumors as an alternative to frozen tissue for gene polymorphism characterization.
- To assess DNA yield, quality, and genotyping success rates from FFPET versus frozen samples.
Main Methods:
- DNA isolation from 50 pediatric central nervous system tumor frozen biopsies and matched FFPET samples.
- Real-time polymerase chain reaction (PCR) for DNA quantification and genotyping of GSTT1, GSTM1, GSTP1, and MTHFR gene polymorphisms.
- Investigation of whole-genome amplification (WGA) to enhance DNA yield from FFPET.
Main Results:
- DNA from FFPET samples showed lower yield and increased fragmentation compared to frozen samples.
- Whole-genome amplification (WGA) did not improve DNA yield from FFPET.
- Successful genotyping (98% concordance) for GSTP1 and MTHFR polymorphisms from FFPET DNA.
- Poor performance in real-time PCR assays for GSTM1 and GSTT1 deletion polymorphisms using FFPET DNA.
Conclusions:
- FFPET DNA is fragmented and challenging to amplify using WGA, impacting its utility for certain genetic analyses.
- Careful validation of PCR assays is crucial when using FFPET DNA due to variable amplification.
- FFPET DNA can be suitable for specific gene polymorphism genotyping, but limitations exist for others.
