Authors: Ken Dixon, Jack Davis, DeLayney Anderson, Mackenzie Burr, Peyton Worley, Isaac Packer, Bridger Kearns, Jeffrey Okojie
Mentors: Jared Barrott
Insitution: Brigham Young University
Introduction
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 all testing of tumor tissue in clinical cancer care comes almost universally 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 cryopreserved 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.
Methods
Human (n = 48) and murine tissues (n = 10) were processed by traditional formalin fixation and paraffin embedding or placed in cryovials containing HypoThermosol solution. 19 human-matched samples were included. These cryovials were cooled to -80°C slowly and stored in liquid nitrogen until the time of the study. DNA was extracted using the same protocol for both tissue types except that tissues embedded in paraffin were first dewaxed using a xylene substitute followed by a multistep rehydration protocol using ethanol and water. Samples were weighed and calibrated to have the same starting mass. After the column purification, samples were eluted in 20 µL and concentration and purity were measured on a Nanodrop. Purity was determined by calculating the 260/280 ratio. DNA fragment length was measured on an Agilent Fragment Microelectrophoresis Analyzer.
Results
Graded amounts of tumor tissue (5- >50 mg) were used to determine the lowest starting material needed to extract 40 ng/mg of DNA. The average for both sample types reached the minimal threshold of 40 ng/mg. However, 74% of FFPE specimens failed to meet the minimum 40 ng/mg, whereas only 21% were below the threshold in the cryopreserved samples (Figure 1). 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 range was 1.09-2.13 with a mean of 1.79. The 260/280 ratio range in FFPE tissues was 0.85-2.76 with a mean of 1.65 (Figure 2). 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 (Figure 3). 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 (Figure 4).
Conclusions
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.