Multi-omics discovery and clinical validation of COL1A2 alternative splicing isoforms as liquid biopsy markers for metastasis surveillance in osteosarcoma.

Abstract

3055 Background: Osteosarcoma (OS) is characterized by early metastatic dissemination, yet the lack of sensitive and tumor-specific tools for real-time surveillance remains a major barrier to improving patient outcomes. Methods: We performed an integrative multi-omics analysis of 424 OS patients, incorporating whole-exome sequencing, bulk RNA sequencing, short-read single-cell RNA sequencing, and single-cell long-read transcriptomics to resolve isoform-level AS alterations at single-cell resolution. Candidate isoforms were evaluated across plasma and peripheral blood datasets from 346 healthy controls to assess tumor specificity. Translational utility was tested in a prospective clinical trial (ChiCTR2400079438), comparing COL1A2 isoform–based circulating tumor cells (CTCs) detection with conventional VIM/TWIST1-based CTC assays and tumor-informed circulating tumor DNA (ctDNA) profiling. Results: OS exhibited widespread alternative splicing dysregulation across both tumor and stromal compartments, with extensive isoform remodeling revealed by single-cell long-read transcriptomics. Two metastasis-associated COL1A2 isoforms were detected in 98% of OS tumor cells but were entirely absent in healthy controls, demonstrating high tumor specificity. COL1A2 isoform–based CTCs detection significantly outperformed VIM- or TWIST1-based assays for predicting metastasis (AUC 0.833 vs. 0.531, P < 0.01) and achieved accuracy comparable to circulating tumor DNA (ctDNA) assays (AUC 0.833 vs. 0.861, P = 0.78). Notably, COL1A2 isoform–based CTCs detected metastatic progression 5.11 months earlier than conventional imaging, approximately two months earlier than ctDNA. Longitudinal analyses further revealed frequent loss of primary tumor–derived mutations during metastatic evolution, limiting the sensitivity of tumor-informed ctDNA assays, whereas COL1A2 isoform–based CTC detection remained robust. Conclusions: Collectively, this study provides the first systematic comparison of CTCs- and ctDNA-based surveillance strategies in OS and establishes COL1A2 isoform-based CTCs detection as a sensitive, specific, and mutation-agnostic liquid biopsy strategy. Clinical trial information: ChiCTR2400079438.