Extracellular vesicles (EVs), including exosomes, microvesicles, and apoptotic bodies, are small particles released by cells into bodily fluids like blood, urine, and saliva. A liquid biopsy using EVs is a promising and less invasive method for detecting cancer. These vesicles carry molecular information from their cell of origin, including proteins, lipids, RNA, and DNA, making them valuable biomarkers for various diseases, including cancer.

We have previously identified a group of proteins with quantitatively cancer-specific high expression in plasma EVs of cancer patients for the purpose of enabling liquid biopsy of renal or colorectal cancer (Int J Cancer (2018);142:607)(Mol Cancer Res (2021);19:834). However, in many cases, these biomarker candidates were also found to be expressed in several normal organs, and their cancer specificity was insufficient.

Therefore in this study, we established an original proteogenomics analysis method and developed a cancer liquid biopsy technology targeting proteins with cancer-specific somatic mutations, which are expected to have 100% cancer specificity.

Bladder cancer is a cancer with a high risk of recurrence and requires regular follow-up using cystoscopy and contrast-enhanced CT after endoscopic resection. In this study, we aimed to develop a new liquid biopsy technique by detecting mutated proteins in urinary extracellular vesicles as a new, minimally invasive method of diagnosing recurrence.

Seven patients with urothelial carcinoma who underwent transurethral resection of bladder tumor at the University of Tokyo Hospital were included in the study. Genomic DNA was extracted from the resected tumors and blood samples from the patients, and somatic mutations were identified by whole exon sequencing. As the result, 55-444 nonsynonymous mutations and 5-16 frameshift mutations were identified.

Based on this information, the personalized proteogenomic analysis of bladder cancer tissue was performed, resulting in identification of 43 mutated proteins. More importantly, analysis of the patients" urinary EVs revealed 4 mutated proteins in common with tissues.

An absolute quantification system using stable isotope-labeled peptide standards was established for these urinary EV mutated proteins, and the quantitative changes in the patient's treatment prognosis with disease progression were also monitored.

In conclusion, we introduce the potential of EV mutant proteins as a new cancer liquid biopsy modality.