Abstract
PURPOSE
Thin-cut formalin-fixed paraffin-embedded (FFPE) tumor sections on glass slides represent a large, underused biorepository for precision oncology, but their long-term room temperature storage raises concerns about nucleic acid integrity and analytical validity. We evaluated whether 20- to 30-year-old archival breast cancer FFPE slides could support contemporary DNA and RNA molecular profiling, including tumor subtyping using orthogonal DNA, and RNA-based approaches.
METHODS
Two archival cohorts were studied: (1) SWOG S8897 low-risk breast tumors stored as 6-µm unstained FFPE sections on slides for approximately 20-30 years (20 tumors; 19 matched cancer-free lymph nodes [LN]) and (2) similarly aged unrelated FFPE tumor blocks (n = 13) used for assay development. DNA and RNA were extracted with quality assurance/quality control. DNA profiling included low/ultra-low-pass whole genome sequencing for copy number analysis (CNA) and IntClust subtyping plus high-depth targeted sequencing using a 93-gene Unique Molecular Identifier (UMI)–based panel. RNA profiling included RNA-seq on block-derived specimens and a custom droplet digital polymerase chain reaction (ddPCR)–based reverse transcription-quantitative PCR (RT-qPCR) assay to enable subtype calling from highly fragmented RNA.
RESULTS
DNA and RNA were recovered from >90% of specimens, despite extensive fragmentation (RNA in a range of 30-100 nt). RNA Access/Exome and NanoString approaches were unsuccessful in the most degraded slide-derived RNAs, prompting development of the ddPCR-based RT-qPCR assay, which successfully quantified targets and enabled intrinsic-like subtype calling across archival tumors. DNA profiling achieved high success for CNA/subtyping and UMI-based mutation detection, and the dual RNA/DNA framework showed strong subtype concordance (ddPCR
v
IntClust approximately 87% among evaluable cases).
CONCLUSION
Decades-old FFPE slide sections, stored at room temperature with no special conditions, can yield usable DNA along with highly degraded RNA for integrated multiomic molecular profiling and breast cancer subtyping. This capability expands access to legacy trial materials with long-term follow-up and supports efficient retrospective and virtual clinical trials.