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.