Year: 2017
Type: article
Abstract: Next-generation sequencing (NGS) is a robust method to assess clinically relevant gene mutations in tumor specimens; however, this technology requires higher minimum DNA inputs for successful analysis than other molecular techniques. Simultaneously, smaller radiologic targets and image-guided brushing/biopsy techniques lead to smaller sample volumes, while competing ancillary procedures such as rapid onsite evaluation or immunohistochemistry may deplete the amount of tumor available for testing. Delay or cancellation of molecular testing related to inadequate DNA is observed in ∼10% of processed samples in our lab. To minimize this impact, we examined the use of stained cytology samples for molecular testing. Autopsy tissue was used to create essentially identical cytologic preparations stained with Diff-Quik, Papanicolaou, and H&E to be compared to both fresh and formalin-fixed paraffin-embedded (FFPE) tissue. Several organs representing the spectrum of cellularity were sampled from each autopsy (lung, liver, spleen, and thyroid). Two cases with tumor tissue were evaluated in addition to these normal tissue types. Aperio image scanning was utilized to simulate archiving of slides and test the correlation of automated cell counting with the DNA quantity obtained. Coverslips were removed with xylene, imprints were scraped, and DNA extracted using the QIAamp DNA FFPE kit. DNA yield was measured using fluorometry (Qubit), and the functional equivalency of the extracted DNA was assessed by a Syber Green quantitative polymerase chain reaction (qPCR) assay normalized to a standard curve produced from unfixed DNA. NGS using the University of Minnesota Comprehensive Oncology Panel was run on the two tumor slide sets and the precision of results from cytology preps was compared with corresponding FFPE and fresh tissue. The DNA concentration range obtained by Qubit was 1.13-120 ng/µL with the spleen providing highest DNA content and the remaining organs having similar lower but actionable amounts. Pap stain consistently exhibited less DNA concentration compared to cytologic H&E, and Diff-Quik was intermediate. The pixel intensity algorithm used for automated cell counts proved not to be reproducible or predictive of the DNA quantity. No significant preliminary differences were identified in the quality (functional amplification) of DNA as measured by qPCR within the tumor subsets between different stain preparations. Importantly, polymorphisms and tumor driver mutations were reproducible across all three stain preparations and comparable to that seen on the fresh tissue (preliminary concordance of 100% within the filtered variant call set). The initial data demonstrate the reproducible nature of extracting high-quality DNA from several types of cytologic stain preparations across sample types with variable cellularity. This data support the use of cytology samples (including rapid onsite evaluation preps) in the molecular lab when FFPE is inadequate.
Authors: David Cartwright, Bharat Thyagarajan, Sophia Yohe, Andrew Nelson
Institution: University of Minnesota
Language: English
Subfield: Cancer Research
Domain: Life Sciences
Open Access status: bronze
OpenAlex ID: W2592399282