Investigating the association of blood-to-tissue tumor mutation burden (TMB) ratio with overall survival and intratumor heterogeneity (ITH) in advanced NSCLC.

2565 Background: High tumor mutation burden (TMB) measured via tissue-based next-generation sequencing (NGS) (tTMB) has been shown to predict better survival outcomes in certain cancers treated with immune checkpoint inhibitors (ICIs). With the increasing use and sensitivity of blood-based NGS, blood-based TMB (bTMB) is frequently employed as an alternative. However, the prognostic significance of bTMB relative to tTMB and its correlation with intratumoral heterogeneity remain poorly understood. Methods: This study included advanced-stage NSCLC patients who underwent both pre-treatment blood-based NGS (collected between October 2020 and February 2024) and tissue-based NGS. Intratumoral heterogeneity (ITH) was assessed using the mutant-allele tumor heterogeneity (MATH) approach, with blood based NGS (bMATH). The highest allele frequency (HAF) was obtained from blood-based NGS. Survival analyses were conducted using Kaplan-Meier curves and Cox proportional hazards models, focusing on the bTMB/tTMB ratio. Results: A total of 102 patients met the inclusion criteria, 55 of whom had complete data for bTMB, tTMB, bMATH, and HAF. The median follow-up time was 12 months. Treatment regimens included ICI combined with chemotherapy (19 patients), ICIs alone (15), targeted therapy (16), and cytotoxic chemotherapy (5), with 33 receiving first-line treatment and 22 receiving second-line or beyond. The median interval from blood NGS to treatment initiation was 20 days (IQR 8–28), and from tissue NGS to treatment initiation was 65 days (IQR 28–255). Multivariable Cox proportional hazards analysis—adjusting for gender, smoking status, stage, ECOG, regimen type, line of therapy, and numeric values of bMATH, bTMB, tTMB, and the bTMB/tTMB ratio—revealed that a higher bTMB/tTMB ratio was independently associated with inferior overall survival (OS) (HR 1.16, 95% CI 1.03–1.30, p=0.01) but showed no significant difference in progression-free survival (PFS) (HR 1.10, 95% CI 0.94–1.29, p=0.23). The cutoff for the bTMB/tTMB ratio that best stratified OS was 0.81. Patients below this cutoff experienced significantly longer OS (median OS 10 vs. 49 months, HR 0.23, 95% CI 0.05–0.96, p=0.02). Additionally, a moderate positive correlation was observed between bMATH and the bTMB/tTMB ratio (Spearman’s r=0.33, p=0.02). Conclusions: A higher bTMB/tTMB ratio was associated with poorer overall survival in advanced-stage NSCLC, highlighting its potential prognostic value. Moreover, the moderate correlation between bMATH and bTMB/tTMB suggests an interplay between TMB and intratumor heterogeneity. Future studies are needed to confirm these findings and to explore their potential therapeutic implications.