Application of Reference Standards to FFPE
A major drawback of traditionally used controls has been applicability to formalin-fixed paraffin-embedded (FFPE) formats—an important source of material both in clinical and biobank settings. The availability of reference materials representing DNA from FFPE extraction is critical to enable end users to benchmark the effectiveness of their extraction process, and to qualify the extraction potential of materials that may have degraded over time or have undergone suboptimal fixation processes, as well as giving a strong indication of the integrity of the laboratory workflow as a whole.
Horizon Diagnostics’ reference standards have been developed with this requirement in mind, and are available in an FFPE format, containing defined DNA quantities and mutant allelic frequencies. To validate the usefulness of the reference standards for FFPE, a study was carried out using X-MAN™ cell lines with B-Raf, EGFR, K-Ras, and PI3Kα mutations knocked-in to one allele of the corresponding endogenous locus (Figure 2).
To confirm the ploidy of targeted genes within each X-MAN cell line, Bio-Rad’s Droplet Digital™ PCR platform was used, using gene-specific copy-number variation (CNV) assays run in duplex with an RNase P reference gene assay. For B-Raf V600E, three cell lines were created on different parental backgrounds each with different B-Raf copy numbers. In addition, analysis of the V600E allelic frequency in each line was found to be consistent with the expected ploidy.
Each X-MAN cell line was then cultivated, FFPE processed, and sections prepared. For the low-allelic burden FFPE reference standards, a mixture of the mutant and wild-type X-MAN cell-line pairs was prepared prior to fixing and FFPE processing. The final allelic frequencies following DNA extraction were measured using Droplet Digital PCR.
In order to assess FFPE block-to-block consistency, digital images of sections from each FFPE block were hematoxylin and eosin stained before scanning using an aperio® system, with the density and evenness of staining across each section indicating a consistent and homogeneous distribution of cells both within and between blocks.
To more accurately determine inter-block consistency, total recoverable DNA was measured from four sections taken from throughout the FFPE block. DNA yield was consistent both between sections taken from within the same block and between different blocks, demonstrating a high level of consistency between each block.
To assess FFPE block allelic frequencies, the allelic burden of three sections from each FFPE block were analyzed using Droplet Digital PCR. The results confirmed the expected allelic frequencies for each FFPE block, and the consistency between each section highlighted the high degree of homogeneity even at low allele burdens (e.g., 1.4% B-Raf V600E).
In summary, a total of 35 FFPE blocks covering 17 genotypes containing a range of allelic frequencies from 1% to 50% were generated. All blocks demonstrated a high degree of intra- and inter-block consistency, with the Droplet Digital PCR platform clearly distinguishing differences between diploid, triploid, and tetraploid cell lines as well as detecting the allelic frequencies down to 1% across a range of clinically relevant mutations. These highly consistent results confirmed the usefulness of Horizon Diagnostics’ FFPE blocks as reference standards for molecular assay workflows.