Low-coverage whole genome sequencing of FFPE-derived-DNA and extracellular vesicle-associated DNA in patients with metastatic cancer.
e14523 Background: Low-coverage whole genome sequencing (LC-WGS) of tumors provides valuable insight into molecular changes driving oncogenesis. A novel liquid biopsy source of tumor DNA for analysis is from extracellular vesicles (EV) obtained from blood. This study compared copy number alteration (CNA) profiles generated from LC-WGS of Formalin-Fixed Paraffin-Embedded (FFPE) DNA and EV-associated DNA in cancer patients. Methods: Three metastatic base of tongue (BOT), two of which were Human Papillomavirus-related (HPV+) and two metastatic cutaneous squamous cell carcinoma (cSCC) patients were included. EV were isolated using ultracentrifugation from patients’ plasma. DNA was extracted from FFPE tumor tissue and EV. LC-WGS aiming for 0.5-1X coverage was performed using a validated method. CNA profiles were generated using the QDNAseq package, with gains defined as a log2 ratio ≥0.15 and losses < -0.15. Results: CNA profiles of FFPE samples from BOT patients demonstrated significant variation regardless of HPV status, with a mean of 20 regions containing 125 CNA (amplifications or deletions) per sample. The cSCC FFPE samples demonstrated a mean of 23 regions containing 189 CNA per sample. Overall, EV-associated DNA CNA profiles had limited similarity with primary FFPE. EV-associated DNA showed a lower number of CNA regions and CNA, with a mean of 3.7 CNA regions and 11.4 CNA for BOT samples and 6.5 regions and 15.9 CNA for cSCC samples. The HPV-BOT sample showed 2/3 EV-CNA regions matched corresponding FFPE-CNA profile (n = 15). The two HPV+ samples were less consistent with only 1/3 and 1/5 EV-CNA regions matching FFPE-CNA profiles (n = 39 and n = 6, respectively). The two cSCC cases showed more consistency with 4/6 and 6/7 EV-CNA regions matching FFPE-CNA profiles (n = 21 and n = 25, respectively). In the matched CNA regions, the mean CNA in EV-associated DNA was 7.5 and in FFPE-DNA was 97. Conclusions: Although selected EV-associated DNA CNA regions reflected the primary tumors, these were limited in number and did not globally reflect the FFPE derived CNA profiles.