Abstract 4957: DNA quality control metrics in cryopreserved tissue outperform DNA extracted from FFPE tissue
Authors: Barrott Jared James , O'Neal Nikole , Okojie Jeffrey , Booker David , Dixon Ken 
Published: March 2024

Abstract
Personalized cancer care requires molecular characterization of neoplasms. While the research community accepts frozen tissues as the gold standard analyte for molecular assays, the source of tissue for testing of tumor tissue in clinical cancer care comes primarily from formalin-fixed, paraffin-embedded tissue (FFPE). Specific to genomics assays, numerous studies have shown significant discordance in genetic information obtained from FFPE samples and frozen samples. To explain the discordance between FFPE samples and cryopreserved samples, a head-to-head comparison between FFPE and cryopreserved tissues was performed to analyze the DNA yield, DNA purity and DNA quality in terms of DNA length. Additionally, short-read sequencing was performed on matched samples and quality control metrics were compared between the two sample types. Matched human tumors (n = 50) were processed either by formalin fixation and paraffin embedding or placed in cryovials containing cryopreservation medium. These cryovials were cooled slowly to -80°C and stored in liquid nitrogen. DNA was extracted using the same protocol for both tissue types with the exception that tissues embedded in paraffin were first dewaxed followed by a multistep rehydration protocol. Samples were weighed and calibrated for mass. After the column purification and elution, sample concentration and purity were measured. DNA fragment length was measured on a fragment microelectrophoresis analyzer. Graded amounts of tumor tissue were used to determine the lowest starting material needed to extract 200 ng of DNA. The average of all the samples reached the minimal threshold of 200 ng. However,74% of FFPE specimens failed to meet the minimum 40 ng/mg, whereas only 21% were below the threshold in the cryopreserved samples. In the cryopreserved group, the average DNA yield was 222.1 ng/mg, whereas 52.8 ng/mg was obtained from FFPE tissue. For DNA purity in cryopreserved tissues, the 260/280 ratio was 1.76, and in FFPE tissues the mean was 1.78. The DNA Quality Number (DQN) is a measurement of DNA fragment length and the percentage that exceeds the threshold of 300 bp. For FFPE, the DQN was 4.4 compared to a DQN of 9.8 for the cryopreserved samples. Setting a higher threshold of DNA length to 40,000 bp and measuring the area under the curve (AUC), it was observed that cryopreserved samples were 9-fold higher in fragments greater than 40,000 bp. Cryopreserved cancer tissue provides superior quality assurance measurements of DNA over FFFPE. Treatment decisions based on molecular results demand accuracy and validity. The pathology community should support efforts to cryopreserve cancer biospecimens in the clinical setting to provide valid molecular testing results. The automatic pickling of tumor specimens in formalin is no longer an acceptable default.
Citation Format: Jared James Barrott, Nikole O'Neal, Jeffrey Okojie, David Booker, Ken Dixon. DNA quality control metrics in cryopreserved tissue outperform DNA extracted from FFPE tissue [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4957.