Molecular profiling of neoadjuvant immunochemotherapy and identification of residual cancer cells in pCR NSCLC: A single-cell analysis of CTONG 1804 clinical trial.

8025 Background: Previously, we reported the clinical findings of stage-one enrollment from a phase II trial of neoadjuvant immunochemotherapy (IO) in untreated patients with resectable non-small cell lung cancer (NSCLC) (CTONG1804, NCT04015778). Recently, two-stage enrollment has been completed. This trial provided an opportunity to investigate the correlation of pathological response and early immune microenvironment during neoadjuvant IO. Methods: We conducted single-cell RNA sequencing (scRNA-seq) on fresh tumor tissue of 21 patients at pre- and post-IO treatment. Multi-omics sequencing was also used in this exploratory study, that included bulk RNA sequencing and tumor-informed MRD sequencing. Results: The pathological complete response (pCR) rate was 42.9% (9/21). Unexpectedly, a total of 143 cancer cells with genome alterations were identified in six (6/9=66.7%) patients with pCR. Only one pCR patient presented MRD positive within one month after surgery, who had the highest number of cancer cells. These residual cancer cells exhibited reduced proliferative capacity and diminished stem cell-like features but retained epithelial-mesenchymal transition (EMT) markers, suggesting metastatic potential and drug resistance. Elevated antigen presentation pathways, particularly involving CD74-MHC class II, were observed in pCR cancer cells, alongside a significant reduction in tumor neoantigen burden. When comparing the immune cells of different pathological response, we found that conventional dendritic cell type 2 (cDC2) emerged as a critical antigen-presenting cell subtype in pCR patients, enhancing T-cell activation and promoting immune response. Reduced CD4-Treg3 populations correlated with improved treatment outcomes, while CD8-MAIT cells exhibited functional plasticity, transitioning from tumor-promoting to tumor-rejecting phenotypes post-therapy. Conclusions: Our study highlights the persistence of residual cancer cells even in pCR patients and identifies key immune cell subsets, such as cDC2 and CD8-MAIT cells, that play pivotal roles in modulating anti-tumor response. These findings provide valuable insights into the mechanisms of immune activation and suppression in NSCLC and suggest potential biomarkers and therapeutic targets for optimizing neoadjuvant immunochemotherapy. Clinical trial information: NCT04015778 .