Author(s): DeLaney Anderson
Mentor(s): Jared Barrott
Institution BYU
Background & aims: Next generation sequencing specifically whole genome sequencing (WGS) plays a key role in precision medicine. Currently, the main source of cancer biospecimens for WGS is formalin-fixed, paraffin-embedded (FFPE) tissues. This is partly because cryopreserved (CP) tissues have not been routinely stored in real-world clinical settings. This study aimed to compare the outcomes of WGS by using matched CP and FFPE samples. We hypothesized that CP samples would demonstrate superior quality metrics before and after sequencing the DNA. Methods: A total of 50 matched pairs of CP and FFPE tumor samples were obtained from patients with various types of cancers, primarily representing gynecological cancers. DNA was extracted and quality and quantity were determined. WGS was performed using 200 ng of DNA extracted from each sample. We conducted a comparison between CP and FFPE samples in terms of the sequencing quality control metrics and the number of identified variants. Results: Our samples consisted of 18 ovary, 5 liver, 6 kidney, 7 uterus, 4 colon, and 10 other cancers. Metrics were obtain before sequencing, and it was found that gDNA concentration in ng/ul was significantly higher in CP (85.2) than FFPE (12.5) tissue. Additionally, DIN number (FFPE: 4.7; CP: 8.4) and fragment size in base pairs (FFPE: 444.1; CP: 644.6) were also significantly higher in CP tissue. WGS was then performed and it was found that both mean read depth (FFPE: 34.6; CP: 54.2) and insert size (FFPE: 243.6 CP: 365.7) were significantly higher in CP than FFPE tissues. The percentage mapped over 15x was similar and not statistically significant with FFPE being 99.3% and CP being 99.7%. To determine concordance between the matched samples, 94,147 variants were found in FFPE and 80,036 variants were found in CP. The median point mutation overlap (VAP>=5.0%) was used to find a concordance percentage, with that being 43.5% between FFPE and CP tissues (shared: 51,619; FFPE: 38,690; CP:28,434). Tumer mutation burden was determined on 8 samples with FFPE being 13.7 and CP being 6.4, with a p-value = 0.02. Lastly structural variance was measured with FFPE averaging 21 and CP averaging 38. Conclusion: WGS utilizing CP samples provides superior quality indices compared to FFPE samples. Despite matched samples, there was significant discordance in variants identified in the cancers. Therefore, CP sample collection should be prioritized when performing WGS.