Abstract 719: Assessing the utility of cell-free DNA in identifying prostate cancer and characterizing tumor heterogeneity via targeted, whole exome, and whole genome multi-region sequencing
Authors: Chen Emmalyn , Cario Clinton L. , Leong Lancelote , Lopez Karen , Márquez César , Chu Carissa , Li Patricia S. , Oropeza Erica , Tenggara Imelda , Cowan Janet , Simko Jeffry P. , Wells Daniel K. , Kageyama Robin , Chan June M. , Friedlander Terence , Aggarwal Rahul , Feng Felix , Paris Pamela L. , Carroll Peter R. , Witte John S. 
Published: September 2020

Abstract
Early cancer diagnosis, especially while the disease is localized and before symptoms appear, results in significantly higher survival rates compared to late-stage diagnosis. At the time of diagnosis, it is also common to find multiple foci within the prostate in men with localized disease. Cell-free DNA (cfDNA) are short DNA fragments released into circulation by dying cells, which may reflect underlying disease biology and simultaneously allow for the identification of genetically distinct tumor subclones. The objectives of this study are to determine if cfDNA concentration can be used to distinguish between healthy individuals from patients with localized or metastatic castration-resistant prostate cancer (mCRPC), and if somatic mutations identified in tumor tissue are detectable in cfDNA.
This study included samples from 277 individuals: 41 healthy donors, 112 patients with localized prostate cancer who underwent radical prostatectomy (RP) at UCSF, and 124 mCRPC patients. Whole peripheral blood was collected in EDTA tubes or PAXgene Blood ccfDNA tubes. Blood and matched tissue from adjacent normal seminal vesicles and multiple tumor regions (1-9 samples per patient) were collected from patients undergoing RP. Extraction of cfDNA was performed on double spun plasma using the Qiagen QIAamp Circulating Nucleic Acid Kits. After extraction, the concentration and fragment length distribution were measured with a Bioanalyzer 2100 Instrument. Fifty-seven samples (multiple tumor tissue foci and matched blood) from nine patients with localized disease were subjected to whole exome sequencing, and 22 samples from five patients were subjected to whole genome sequencing. All samples from 14 patients underwent targeted sequencing with a 2.5Mb panel generated via machine learning on TCGA prostate cancer sequence data. Somatic variant calling was performed with Broad Institute's Terra platform (GATK4/MuTect2) for tumor tissue and with the Curio platform for cfDNA to build consensus sequences leveraging duplex unique molecular tags. To estimate tumor fraction in cfDNA, ichorCNA was used to profile low pass whole genome sequence data.
Total cfDNA concentration was able to distinguish between healthy and metastatic (p = 0.001) participants, adjusted for age; and also between localized and metastatic groups (p &lt; 0.001), adjusted for age and PSA. Among the patients with localized disease, cfDNA concentration was not associated with age, PSA, Gleason score, or Decipher Score (a 22 gene metastasis risk-predicting RNA gene expression signature), suggesting the potential for this marker to be independent of factors that are commonly used to assess disease burden and risk of progression. Targeted sequencing of prostate tumors resulted in an average of 16 mutations with a range of 1 to 128 mutations per tissue region. The cfDNA allele frequencies for mutations identified in tumor tissue ranged from 0.5% to 20%. Four patients with variant detection in cfDNA also experienced biochemical recurrence. While copy number analysis identified clonal and potentially subclonal alterations in WGS tumor tissue, no alterations were found in WGS cfDNA. Further analysis of potential factors influencing variant detection in cfDNA (adverse pathology, starting amount of DNA, coverage) will be performed.
Citation Format: Emmalyn Chen, Clinton L. Cario, Lancelote Leong, Karen Lopez, César Márquez, Carissa Chu, Patricia S. Li, Erica Oropeza, Imelda Tenggara, Janet Cowan, Jeffry P. Simko, Daniel K. Wells, Robin Kageyama, June M. Chan, Terence Friedlander, Rahul Aggarwal, Felix Feng, Pamela L. Paris, Peter R. Carroll, John S. Witte. Assessing the utility of cell-free DNA in identifying prostate cancer and characterizing tumor heterogeneity via targeted, whole exome, and whole genome multi-region sequencing [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 719.