Abstract 1595: Detection of tumor-derived DNA in urine cell-free DNA of pre-operative renal cell carcinoma patients
Authors: Lin Selena , Linehan Jennifer A. , Kuang Ruby , Guharaj Selvi , Wilson Timothy G. , Hoon Dave SB 
Published: August 2018

Abstract
The current clinical approaches lack reliable methods (i.e. imaging and serum biomarkers) to identify patients with renal cell carcinoma (RCC) resulting in late detection, where the 5-year survival rate is poor (&lt;8%). Thus, there is an urgent need for better minimally-invasive screening and diagnostic blood/urine assays for earlier detection. Given the logistical ease of collecting urine, urinary cell-free DNA (cfDNA) represents an advantageous source to detect tumor biomarkers and permit for routine/repeat sampling. We previously demonstrated urine cfDNA contains tumor-derived DNA mutations in liver cancer, distal of the kidney filtration barrier. However to date, urinary cfDNA as a source of RCC biomarkers remains unexplored. We hypothesize due to the direct contact of urinary flow-through with the kidney organ that urinary cfDNA is enriched for RCC-derived DNA mutations. To test this, we evaluated the feasibility of detecting tumor-derived DNA mutations utilizing a hotspot 50-cancer gene panel droplet next generation sequencing (dNGS, Raindance Technologies) assay in a pilot cohort of 42 specimens obtained from 13 unique RCC patients and 3 controls (2 RCC cell-lines and 1 normal DNA). The specimens evaluated include matched tumor formalin-fix paraffin-embedded (FPPE) DNA (n=11), urine cfDNA (n=15), and peripheral blood lymphocytes (PBL) DNA (n=13), serving as a germline control. 150ng of genomic DNA from FFPE or PBLs and ≥5ng of urine cfDNA was used for the dNGS assay. An average of 5.5E10 reads were obtained of which 86% were on-target for all 42 specimens providing an average 3.9E3x coverage. Initial dNGS variant analysis utilizing a conservative 5% variant frequency (VF%) cut-off was performed in 11 unique RCC patients containing matched FFPE-urine cfDNA-PBL, of which five patients also contained post-operative urine. 100% of tumor FFPEs (n=11) contained detectable single nucleotide variants (SNVs) (1-10 SNVs) upon germline variant removal. 80% of matched urine cfDNA contained ≥1 tumor-concordant SNV. Additionally, 37.5% of patients contained previously reported somatic mutations (i.e. PIK3CA T1025T, RET L769L, TP53 H233Y, etc) that were tumor-urine concordant. Interestingly, three patients harbored known somatic mutations that were not detected in the matched tumor and three post-operative urine cfDNA specimens contained tumor-derived SNVs despite surgical resection. Ongoing follow-up will reveal if urine cfDNA is a suitable source for identifying tumor heterogeneity and/or residual disease in RCC patients. Furthermore, evaluation of matched plasma is ongoing. Overall, this proof-of-concept study demonstrates feasibility of utilizing urinary cfDNA as a noninvasive source to detect RCC-related biomarkers and provides a potential platform to study and discover novel RCC biomarkers for earlier detection of new/recurrent RCCs.
Citation Format: Selena Lin, Jennifer A. Linehan, Ruby Kuang, Selvi Guharaj, Timothy G. Wilson, Dave SB Hoon. Detection of tumor-derived DNA in urine cell-free DNA of pre-operative renal cell carcinoma patients [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1595.