Abstract 1322: Development and performance of a formalin-damaged multiplexed DNA tumor mutation FFPE reference material
Authors: Lowe Dana J. Ruminski , Philkana Deepika , Huang Catherine , Butler Matthew G. , Clement Omoshile , Anekella Bharathi 
Published: September 2020

Tumor profiling often begins with a tissue biopsy that is formalin fixed and paraffin embedded (FFPE), a process that introduces various kinds of damage in the nucleic acids found in the tissue, however FFPE reference materials that closely mimic the damage profile of patient samples are lacking. Depurination, depyrimidination, deamination, oxidation, nicks, and double strand breaks may be found in DNA extracted from FFPE tissue, despite the use of extraction kits that attempt to repair some of this damage. We have developed a formalin-damaged, multiplexed biosynthetic reference material, Seraseq® FFPE Tumor DNA, to mimic this type of damage found in patient samples to create a more patient-like control. Biosynthetic DNA containing 24 cancer variants of interest were pooled in equivalent concentrations and introduced into the GM24385 reference cell line (Coriell) to simulate SNVs, indels, and structural rearrangements. Full length, allele-specific copies of ERBB2, MET, and MYC were also introduced to create copy number amplifications (CNV). Engineered cells were diluted to achieve desired allele frequencies and copy numbers as determined by digital PCR. The cells were processed with a proprietary FFPE protocol that mimics the damage seen in patient samples. Various FFPE extraction kits were used to isolate DNA and the amount of damage was assessed using the KAPA hgDNA Quantification & QC Kit. Allele frequencies and copy numbers were determined by digital PCR, the Archer VariantPlex Solid Tumor assay, and the Illumina TruSight Tumor 170 assay. DNA extractions of the FFPE reference material with the Qiagen QIAamp DNA FFPE Tissue Kit and the Promega Maxwell RSC FFPE DNA Kit yielded similar amounts (&gt;200 ng per curl). All 24 variants were detected close to the 5-10% targeted allele frequency range and CNVs were within 5-10 total copies by dPCR. Similar results were observed with the TST170 assay. The quality as measured by Q120 bp/Q41 bp ratio using the KAPA hgDNA Quantification & QC Kit of the FFPE extracted damaged DNA was reduced by ~33% compared to non-damaged DNA extracted from FFPE, closer to the quality seen for some patient samples while still maintaining good amplifiability in downstream processes. The Seraseq FFPE Tumor DNA Reference Material performs as designed in digital PCR, amplicon-based, and hybrid capture-based NGS assays. It can be used in full process assay development and validation, bioinformatics pipeline optimization, and as a positive control in clinical research. The biosynthetic manufacturing approach allows for multiplexing of customizable variants and adjustable DNA damage.

Citation Format: Dana J. Ruminski Lowe, Deepika Philkana, Catherine Huang, Matthew G. Butler, Omoshile Clement, Bharathi Anekella. Development and performance of a formalin-damaged multiplexed DNA tumor mutation FFPE reference material [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1322.