At the time of lung cancer diagnosis, approximately half of the patients have existing metastases. In lung adenocarcinoma (LADC), brain metastases are the most common, which have a significant negative impact on patient outcomes. The main objective of this study was to characterize surgically removed primary tumors and brain metastases from patients with LADC to better understand tumor heterogeneity and plasticity during disease progression.

Formalin-fixed and paraffin-embedded primary and brain tumor tissues from 58 patients were subjected to proteomic analysis using a previously established sample preparation protocol (Kuras et al., 2021). Peptides were labeled with TMTpro 16-plex, and the proteomic analysis was performed using high-performance liquid chromatography coupled with tandem mass spectrometry (Ultimate 3000 UPLC, QExactive HF-X). Protein identification and quantification were carried out using Proteome Discoverer 2.4 software. Statistical and bioinformatic analyses were conducted using R and Perseus software.

The proteomic analysis identified 10,862 proteins, from which 7,418 proteins were quantified across all 116 samples. Principal component analysis separated the samples based on tissue origin, revealing a greater heterogeneity among the brain metastases than the primary tumors. Four proteomic subtypes were detected by consensus clustering in both primary tumors and brain metastases, of which the primary subtypes showed an association with progression-free survival (PFS). The primary subtype with worse PFS was characterized by fast proliferation, which was indicated by higher expression of Ki67, Myc targets, and cell-cycle-related pathways. In contrast, the subtype with the best PFS showed higher expression of immune-related proteins. Downregulation of protective immune mechanisms was observed in brain metastases compared to primary tumors. Additionally, there was no significant association between primary and metastatic subtypes, which is indicative of a phenotypic change during disease progression.

Our results provide insight into the molecular heterogeneity of LADC tumors and the development of brain metastasis. Better disease stratification could be obtained by proteomic profiling of resected primary tumors and metastatic lesions. Our results indicate a phenotypic change occurring during disease progression, which is why the effectiveness of metastasis treatments based only on examining primary tumors is uncertain.

Funding: Royal Physiographic Society in Lund, Mrs. Berta Kamprad"s Cancer Foundation (FBKS-2020-22-(291)), National Research, Development and Innovation Office (2021-1.2.4-TÉT-2021-00030).