Molecular Features of Lymph Node Metastasis in T1/2 Colorectal Cancer from Formalin-Fixed Paraffin-Embedded Archival Specimens

Histological risk factors for lymph node metastasis (LNM) in early-stage colorectal cancers (CRC) have been described, although the predictive utility of these factors varies. Improved LNM risk assessment based on findings in endoscopic colon and rectal excisions is necessary for optimal surgical management of CRC patients with pathologic T1- /T2-staged invasive depth (i.e., tumor not invading beyond the muscularis propria layer); as the current system is overly conservative, and results in many unnecessary radical surgeries. To identify molecular features in early CRC with elevated LNM potential, we carried out proteomic and gene expression profiling to compare T1 lymph node (LN) negative with T1/2 LN positive CRC tumors from formalin-fixed paraffin-embedded (FFPE) specimens. Using a data-independent acquisition mass spectrometry workflow, we detected over 7400 proteins and quantified over 4400 in all 21 specimens. Proteins from tumors with LN metastasis were enriched with effectors of epithelial–mesenchymal transition (EMT) and gene expression profiling confirmed activation of key transcription factors, SNAI1 and ZEB1, as well as a reduction in E-cadherin expression. Toward an implementation pathway, we investigated immunohistochemistry assays targeting four EMT-related proteins. While MS could reliably discern twofold protein abundance changes, we found the semiquantitative nature of IHC scoring limited confirmation of this degree of protein expression difference. This study demonstrated that EMT effectors are associated with locoregional metastasis in T1/T2 CRC and could be used to augment metastatic risk assessment, although further developments are required to enable routine implementation.