biostudies-literatureCapitoli GItalian Ministry of HealthItalian Ministry of University and Research (MUR) through the "Future Artificial Intelligence Reseacrh - FAIR" program within the National Recovery and Resilience Plan (PNRR), Mission 4, Component 2, Investment 1.3 - funded by the European Union - NextGenerationEU. Project: "AIDH - Adaptive AI Methods for Digital Health"National Plan for NRRP Complementary InvestmentsItalian Ministry of the University MUR Dipartimenti di Eccellenza 2023-2027 IMPACT Medicine ProjectItalian Ministry of Innovations via the Sustainable Growth Fund - Innovation Agreements800-809https://www.ebi.ac.uk/biostudies/studies/S-EPMC12572012biostudies-literatureUnknown90(2)<h4>Purpose</h4>The identification of novel molecular biomarkers may assist in the characterization of indeterminate thyroid nodules, which pose significant diagnostic challenges. Here, we aimed to explore the potential of proteomic analyses to support biomarker discovery in challenging thyroid lesions.<h4>Methods</h4>Linear Discriminant Analysis (LDA) was applied to Matrix-Assisted Laser Desorption Ionization Mass Spectrometry Imaging (MALDI-MSI) data from 44 thyroid neoplasms to select the most impactful molecular features for the classification of different tumor histologies, as well as for the distinction between NRAS-mutant (mNRAS) and NRAS-wild-type (wtNRAS) tumors. Relevant peaks were subsequently identified through nanoscale liquid chromatography electrospray ionization tandem mass spectrometry (nLC-ESI-MS/MS).<h4>Results</h4>The LDA selected nine relevant molecular markers distinguishing noninvasive follicular thyroid neoplasms with papillary-like nuclear features (NIFTPs) from other tumor histologies (balanced accuracy = 73%), as well as 19 relevant markers able to identify mNRAS cases (balanced accuracy = 84%). Nine differentially expressed proteins were putatively identified: among them, ATP-dependent RNA helicase DDX42 showed a similar distribution between NIFTPs and papillary thyroid carcinomas (PTCs) / follicular variant PTCs (FVPTCs), while the distribution of the Histone H4 signal was similar between NIFTPs and follicular adenomas (FAs). In addition, Protein disulfide-isomerase A1 and Complement C4-B were overexpressed in wtNRAS compared to mNRAS cases, regardless of histology.<h4>Conclusion</h4>The LDA-selected features enable to distinguish NIFTPs from morphologically similar lesions and to discriminate between mNRAS and wtNRAS cases. The identified markers might complement genetic analyses and provide insights into the distinct pathogenic drivers behind the development of mNRAS compared to wtNRAS lesions.EndocrineBiomarker identification through spatial proteomics for the characterization of indeterminate thyroid nodules.PMC12572012IMPACT MEDICINE, I. 232/2016, art. 1, commi 314-337PE0000013, CUP D53C22002380006Ricerca Corrente 5x1000F/350104/01-02/X60PNC0000003 - AdvaNced Technologies for Human-centrEd Medicine (project acronym: ANTHEM)Fusco NPagni FPiga IGreco AMaggioni ALeni DAlviano AMCapitoli GMagni FMonza NGatti AVGalimberti SGarancini MPagani LMaffini FDenti VBernasconi DPL'Imperio VfalseBiomarker identification through spatial proteomics for the characterization of indeterminate thyroid nodules.<h4>Purpose</h4>The identification of novel molecular biomarkers may assist in the characterization of indeterminate thyroid nodules, which pose significant diagnostic challenges. Here, we aimed to explore the potential of proteomic analyses to support biomarker discovery in challenging thyroid lesions.<h4>Methods</h4>Linear Discriminant Analysis (LDA) was applied to Matrix-Assisted Laser Desorption Ionization Mass Spectrometry Imaging (MALDI-MSI) data from 44 thyroid neoplasms to select the most impactful molecular features for the classification of different tumor histologies, as well as for the distinction between NRAS-mutant (mNRAS) and NRAS-wild-type (wtNRAS) tumors. Relevant peaks were subsequently identified through nanoscale liquid chromatography electrospray ionization tandem mass spectrometry (nLC-ESI-MS/MS).<h4>Results</h4>The LDA selected nine relevant molecular markers distinguishing noninvasive follicular thyroid neoplasms with papillary-like nuclear features (NIFTPs) from other tumor histologies (balanced accuracy = 73%), as well as 19 relevant markers able to identify mNRAS cases (balanced accuracy = 84%). Nine differentially expressed proteins were putatively identified: among them, ATP-dependent RNA helicase DDX42 showed a similar distribution between NIFTPs and papillary thyroid carcinomas (PTCs) / follicular variant PTCs (FVPTCs), while the distribution of the Histone H4 signal was similar between NIFTPs and follicular adenomas (FAs). In addition, Protein disulfide-isomerase A1 and Complement C4-B were overexpressed in wtNRAS compared to mNRAS cases, regardless of histology.<h4>Conclusion</h4>The LDA-selected features enable to distinguish NIFTPs from morphologically similar lesions and to discriminate between mNRAS and wtNRAS cases. The identified markers might complement genetic analyses and provide insights into the distinct pathogenic drivers behind the development of mNRAS compared to wtNRAS lesions.2025-01-01T00:00:00Z2025 Nov2026-06-12T05:03:05.405Z2026-06-12T03:07:41.581ZS-EPMC125720124079009910.1007/s12020-025-04383-9