Introduction to Molecular Data and FFPE Tissue Blocks
Formalin fixed paraffin embedded (FFPE) tissue blocks are now widely used in all fields of study. The routine processing of tissues from post-mortem, surgery, and biopsy has led to a vast collection of specimens that represent various diseases and its different stages. Many tumor or normal tissue slides are screened by a pathologist to look for biomarkers related to cancer. This makes these specimens more attractive in the identification of molecular changes that are linked with the onset and progression of cancer. However, the quality of DNA and RNA that are extracted from FFPE tissue blocks are lower compared to those from fresh or frozen specimens due to degradation and lower essay efficacy. The degradation can occur due to sub optimal fixation, non-standardized processing and extraction methods. Not all FFPE tissue blocks are equal as it can be affected by factors such as:
- Biospecimen size
- pH of formalin
- Composition of formalin
- Temperature of fixation
- Duration of fixation
- Tissue block or slide storage
The yield and quality of extracted DNA and RNA are also affected by sub optimal methods of extraction. Although there are many challenges, the data generated from the genomic and gene expression of FFPE tissue blocks are accepted as an accurate reflection of the patient’s condition.
a) Success rates and challenges of DNA analysis in FFPE tissue blocks
FFPE tissue block analysis can be used to evaluate the genetics and pathology of these specimens which can lead to a better understanding of how genes play a role in the progression of cancer and other diseases. The success rates using FFPE tissue blocks are lower compared to fresh or frozen specimens.
b) Accuracy of FFPE tissue blocks in genotyping
Genotype concordance between frozen and FFPE specimens varies depending on analytical platforms used. Although the values observed support that FFPE tissue blocks should be used in genotype determination, these values are unable to capture the differences in copy number or the rates of false discovery in cancer related mutations. The big differences in rates of false discovery in cancer related mutations have resulted in warnings against using FFPR tissue blocks without proper validation. The discordance of genotyping results between frozen and FFPE specimens have been thought to be due to the increase in number of mutations found in FFPE tissue blocks. Specifically, it as observed that there were higher numbers of transversion mutations, small insertions or deletions, and transition mutations in FFPE blocks. In single nucleotide polymorphism using microarray, FFPE tissue blocks were found to be the most successful with lowest miscall counts and loss of detection.
c) Accuracy in copy number determination
Many studies contradict whether the copy number is similar between frozen and FFPE specimens. The largest differences between frozen and FFPE specimens were seen in whole genome amplification. A contributing factor in the copy number disparity between frozen and FFPE specimens is tumor heterogeneity.
d) Accuracy of FFPE in DNA methylation
The accuracy of methylation data using FFPE specimens depends how it is used.
e) DNA analysis of FFPE tissue blocks
The accuracy of copy number determination and genotyping using FFPE specimens vary according to analytical platforms and publications. However, there is a high degree of agreement using frozen specimens for genotyping while FFPE specimens are analyzed by microarray. Disagreement for copy number analysis can be minimized by omitting whole genome amplification and using oligonucleotide arrays instead of single nucleotide polymorphism arrays. In DNA methylation analysis, there was improvement in detection rates when DNA was extracted from FFPE tissue blocks using a kit specific for FFPE.
f) RNA expression analysis
RT-PCR and transcriptome analysis in FFPE tissue blocks would help in the rapid screening and confirmation of transcripts that are involved in the cause and progression of disease. However, the success rates for these analytical platforms differ when FFPE tissue blocks are used.
g) Accuracy of RT-PCR in FFPE specimens
In FFPE specimens, the accuracy of RNA expression depends on analytical factors such as RNA amplification, transcript of interest, or the number of genes utilized in normalization. Comparing FFPE and froze specimens, the differences in relative expression can be influenced by the number of transcripts utilized in normalization of real time qRT-PCR.
h) Microarray and DASL
RNA expression data accuracy produced by FFPE specimens through microarray or DASL varies with RNA subtypes. The discordance between frozen and FFPE specimens can be attributed to the location of the probe in the transcript. Probes targeting the 5’ end of the transcript in FFPE specimens have a less intense signal compared to the ones found in the 3’ untranslated region.
i) RNA analysis in FFPE specimens
Conducting mRNA transcriptome analysis in FFPE specimens can lead to artifactual findings unless authentication studies are made using frozen or fresh specimens.
Greytak SR, Engel KB, Bass BP, Moore HM. Accuracy of molecular data generated with FFPE biospecimens: lessons from the literature. Cancer Res. 2015; 75(8):1541-1547.