Liquid Biopsies Using Plasma Exosomal Nucleic Acids and Plasma Cell-Free DNA Compared with Clinical Outcomes of Patients with Advanced Cancers

Abstract

Purpose: Blood-based liquid biopsies offer easy access to genomic material for molecular diagnostics in cancer. Commonly used cell-free DNA (cfDNA) originates from dying cells. Exosomal nucleic acids (exoNAs) originate from living cells, which can better reflect underlying cancer biology.Experimental Design: Next-generation sequencing (NGS) was used to test exoNA, and droplet digital PCR (ddPCR) and BEAMing PCR were used to test cfDNA for BRAF^V600, KRAS^G12/G13, and EGFR^{exon19del/L858R} mutations in 43 patients with progressing advanced cancers. Results were compared with clinical testing of archival tumor tissue and clinical outcomes.Results: Forty-one patients had BRAF, KRAS, or EGFR mutations in tumor tissue. These mutations were detected by NGS in 95% of plasma exoNA samples, by ddPCR in 92% of cfDNA samples, and by BEAMing in 97% cfDNA samples. NGS of exoNA did not detect any mutations not present in tumor, whereas ddPCR and BEAMing detected one and two such mutations, respectively. Compared with patients with high exoNA mutation allelic frequency (MAF), patients with low MAF had longer median survival (11.8 vs. 5.9 months; P = 0.006) and time to treatment failure (7.4 vs. 2.3 months; P = 0.009). A low amount of exoNA was associated with partial response and stable disease ≥6 months (P = 0.006).Conclusions: NGS of plasma exoNA for common BRAF, KRAS, and EGFR mutations has high sensitivity compared with clinical testing of archival tumor and testing of plasma cfDNA. Low exoNA MAF is an independent prognostic factor for longer survival. Clin Cancer Res; 24(1); 181-8. ©2017 AACR.

Figure 1

Overall survival (OS) per mutation allelic frequency (MAF) of KRAS, BRAF, or EGFR mutations in plasma. A, Twenty-one patients with a low MAF (≤median; blue dashed line) in plasma exoNA had a significantly longer median OS duration than 22 patients with a high MAF (>median; red line; 11.8 vs. 5.9 months; P=0.006). B, Twenty-one patients with a low MAF (blue dashed line) in plasma cfDNA tested with ddPCR had a significantly longer median OS than 20 patients with a high MAF (red line; 8.5 vs. 5.9 months; P=0.023). C, There was a trend for 19 patients with a low MAF (blue dashed line) in plasma cfDNA tested with BEAMing to have a longer median OS than 18 patients with a high MAF (red line; 7.4 vs. 6.5 months; P=0.066).

Figure 2

Time to treatment failure (TTF) per pretreatment mutation allelic frequency (MAF) of KRAS, BRAF, or EGFR mutations in plasma. A, Fifteen patients with a low MAF (≤median; blue dashed line) in plasma exoNA had a significantly longer median TTF than 17 patients with a high MAF (>median; red line; 7.4 vs. 2.3 months; P=0.009). B, Fourteen patients with a low MAF (blue dashed line) in plasma cfDNA tested with droplet digital PCR had a significantly longer median TTF than 17 patients with a high MAF (red line; 8.6 vs. 2.3 months; P=0.001). C, Twelve patients with a low MAF (blue dashed line) in plasma cfDNA tested with BEAMing had a trend towards a longer median TTF than 15 patients with a high MAF (red line; 3.7 vs. 2.3 months; P=0.086).

Figure 3

Pretreatment mutation allelic frequency (MAF) of KRAS, BRAF, or EGFR mutations in plasma and corresponding response to systemic therapy per RECIST 1.1. A, Twelve patients with a partial response (PR) or stable disease (SD) ≥6 months had a significantly lower median mutated exoNA MAF than 20 patients with a PD or SD <6 months (0.43% vs. 14.74%; P=0.006. B, Twelve patients with a PR or SD ≥6 months did not have a statistically different median mutated cfDNA MAF (assessed with droplet digital PCR) compared with 19 patients with a PD or SD <6 months (0.70% vs. 16.00%; P=0.24). C, Ten patients with a PR or SD ≥6 months did not have a statistically different median mutated cfDNA MAF (assessed with BEAMing) compared with 17 patients with a PD or SD <6 months (5.44% vs. 12.88%; P=0.24).