The molecular stethoscope: RNA of drug metabolising enzymes in circulating vesicles correlates with their specific protein content in the liver

Background: Precision dosing aims to deliver the right drug dose for a specific patient based on individual characteristics, improving efficacy and reducing toxicity. Multi-omic approaches and ‘liquid biopsy’ assays are expected to facilitate the use of precision dosing by linking an organ, such as liver (site of drug metabolism) to a minimally-invasive biopsy, e.g. blood.  Methods: Liver tissue (20-250 mg) and plasma (1-3 mL) samples from the same cancer patients (n=9) were analysed using proteomic (targeted and global) and transcriptomic (NGS) methods. Healthy controls (n=5) were used as baseline. In-house QconCAT methodology was used for protein quantification on nanoHPLC-Orbitrap Elite system (Thermo) using DDA mode with an inclusion list. NGS followed Ampliseq workflow performed at Life Technologies (Thermo, Texas) at a depth of 8 million reads/sample. Expression levels of drug-metabolising enzymes were normalized by a novel liver-specific shedding correction factor (LSCF), computed using a combination of 14 liver-specific marker genes measured in plasma.  Results: Liver proteins (~2,500) and plasma RNA transcripts (~21,000) were measured in cancer samples and normal controls. Data for key drug-metabolising enzymes (the targets) and 14 marker genes were processed. Coverage of targets and markers was 80-100% in the protein data and 64-100% in NGS data. LSCF was higher and more variable in cancer patients than healthy controls; LSCF_(cancer)=21.62±16.30 rpm, n=9; LSCF_(healthy)=0.83±0.26 rpm, n=5; t-test p<0.01). Tissue protein and LSCF-corrected plasma RNA levels were assessed for correlation; major drug-metabolising enzymes were significantly correlated between plasma (RNA) and liver (protein); CYP3A4 (Pearson r=0.98, p<0.01, R²=0.95); CYP2C9 (r=0.76, p=0.03, R²=0.57); CYP1A2 (r=0.93, p=0.02, R²=0.86); CYP2A6 (r=0.98, p<0.01, R²=0.96).  Conclusion: A liquid biopsy test for non-invasive assessment of liver content of key enzymes in plasma was established; caveats: (a) enzymes exclusively/predominantly expressed in liver and (b) predominantly shed into the blood. This technology should facilitate efforts towards precision dosing/medicine.