Spatially Resolved Molecular Characterization of Noninvasive Follicular Thyroid Neoplasms with Papillary-like Nuclear Features (NIFTPs) Identifies a Distinct Proteomic Signature Associated with RAS-Mutant Lesions

Follicular-patterned thyroid neoplasms comprise a diverse group of lesions that pose significant challenges in terms of differential diagnosis based solely on morphologic and genetic features. Thus, the identification of easily testable biomarkers complementing microscopic and genetic analyses is a highly anticipated advancement that could improve diagnostic accuracy, particularly for noninvasive follicular thyroid neoplasms with papillary-like nuclear features (NIFTPs). These tumors exhibit considerable morphological and molecular heterogeneity, which may complicate their distinction from structurally similar neoplasms, especially when genetic analyses reveal shared genomic alterations (e.g., <i>RAS</i> mutations). Here, we integrated next-generation sequencing (NGS) with matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI) to perform a proteogenomic analysis on 85 NIFTPs (n = 30 <i>RAS</i>-mutant [<i>RAS</i>-mut] and n = 55 <i>RAS</i>-wild type [<i>RAS</i>-wt]), with the aim to detect putative biomarkers of RAS-mut lesions. Through this combined approach, we identified four proteins that were significantly underexpressed in <i>RAS</i>-mut as compared to RAS-wt NIFTPs. These proteins could serve as readily accessible markers in morphologically borderline cases showing <i>RAS</i> mutations. Additionally, our findings may provide insights into the distinct pathogenic pathways through which <i>RAS</i>-mut and <i>RAS</i>-wt NIFTPs arise, highlighting the pivotal role of constitutive RAS-mitogen-activated protein kinase (MAPK) pathway activation in the development and progression of <i>RAS</i>-mut tumors.