Intratumoral heterogeneity as a potential biomarker for immunotherapy response and postoperative recurrence in NSCLC.

Intratumoral heterogeneity (ITH) is associated with poor prognosis in various solid tumors. However, its use as a predictive biomarker for immunotherapy in advanced non-small cell lung cancer (NSCLC) is still lacking. We used Maftools to analyze somatic variants of NSCLC from independent cohorts (POPLAR, OAK, Hellmann2018) including 504 patients with advanced NSCLC. Additionally, 121 patients (in-house cohort) with NSCLC who received radical treatment and underwent 733-panel NGS testing of surgical samples were included. We used Shannon entropy to measure ITH, analyzed correlation with immunogenic markers, and explored ITH's prognostic effect on immunotherapy and its role in predicting postoperative recurrence risk. Using the TCGA cohort, we compared genetic characteristics between low ITH (ITH-L) and high ITH (ITH-H) groups, assessed mutational profiles, and evaluated associations between ITH scores and immune cell populations. In the Hellmann2018 cohort, ITH-L correlated with higher durable clinical benefit (DCB) rate, objective response rate (ORR), and median progression-free survival (mPFS), highlighting ITH-L as a potential positive predictor for ICI therapy. Combining ITH with TMB revealed that ITH-L/TMB-H patients had significantly better DCB, ORR, and mPFS, supporting the potential predictive value of ITH when evaluated together with TMB. Validation in POPLAR/OAK cohorts supported the association of ITH, alone or combined with TMB, with immunotherapy outcomes. ITH-L patients exhibited higher DCB, ORR, longer mPFS, and median overall survival (mOS). Combining ITH and circulating tumor DNA validated these findings. In early-stage surgical NSCLC patients, ITH-L was associated with significantly higher median recurrence-free survival (mRFS). Mechanistically, the ITH-L group showed elevated TMB, neoantigen (SNV), and subclonal genome fraction, alongside reduced HRD scores and CNV burden. Mutational analysis identified XIRP2 as the most frequently altered gene, enriched in the ITH-L group, while SHOX2 was the only gene with higher frequency in the ITH-H group. Immune infiltration analysis revealed significant differences in M1 macrophages, activated memory CD4+ T cells, and naive CD4+ T cells between groups. ITH may serve as a potential biomarker associated with immunotherapy outcomes in advanced NSCLC and postoperative recurrence risk in early-stage surgical patients.