ADVANCES IN CLINICAL PROTEOMICS FOR ANALYSIS OF THYROID FINE NEEDLE ASPIRATION BIOPSIES: EVALUATING PROTEOMIC STABILITY IN PRESERVATIVE SOLUTIONS

Introduction Thyroid nodule lesions is one of the diseases diagnosed using liquid-based cytology [1]. Such samples are collected via fine-needle aspiration biopsy (FNAB) and deposited into preservative solutions in order to maintain their morphological integrity. However, the proteomic stability of these preserved samples is yet to be investigated and must be ascertained in order for them to be reliably employed in proteomic studies aimed at biomarker discovery. Methods Thyroid FNABswere collected from 14 patients (San Gerardo Hospital, Monza, Italy) and transferred into CytoLyt solution, centrifuged and re-suspended in PreservCyt solution. Cytospin spots have been positioned onto ITO-conductive slides and MALDI-MSI intact proteins analysis was performed using an ultrafleXtreme MALDI-TOF/TOF (Bruker Daltonik). Spectra pre-processing and data analysis were performed using the open-source R software v. 3.4.3. Results Each FNAB was split into several samples in order to investigate the experimental repeatability (intra-day and inter-day) of the proteomics analysis and the cytological samples stability after 7, 14 days and 2 months in PreservCyt and after 7 days in CytoLyt at 4°C. All samples were compared with the one prepared at t0. Mass spectra similarity was evaluated by using two score systems. The score S3, derived from a previous study [2],was the sum of three components (fit, retrofit and spearman’s correlation). The second score system (S4) ranges 0–4 and includes a fourth feature that measure the overlap, which takes into account the whole shape of the two spectra. Intra-day and inter-day CV were very low within ranges of 8.64%- 12.03% for S3 and of 7.37%-10.43% for S4, respectively. The results suggest no substantial deviations from t0 when the cytological samples were stored in PreservCyt until 14 days and in CytoLyt until 7days. However longer storage time (2 months) in PreservCyt does not preserve the specimens, and the spectra overlap with t0 was only 50%. Conclusions This study represents a step forward towards the implementation of MALDI-MSI, combined with a trustworthy and robust sample preparation methodology, into the cytopathology routine, integrating the morphology with the proteomics data to improve the diagnosis. Moreover, this protocol allow simple sample collection and shipment to be used not only for the proteomic MALDI-MSI analysis of thyroid FNABs but also for other biological liquid based specimens. Novel Aspect A new similarity score was introduced to equally take into account the number of signals (fit and retrofit) 272 and their intensities (spearman’s correlation and spectra overlap). References 1. Rossi ED, Zannoni GF, Moncelsi S, Stigliano E, et al., Frontiers in Endocrinology, 3, 1-4 (2012). 2. Hollemeyer K, Altmeyer W, Heinzle E, Pitra C, Rapid Communication in Mass Spectrometry, 22, 2751– 2767 (2008). Funding: This work was funded thanks to AIRC (Associazione Italiana per la Ricerca sul Cancro) MFAG GRANT 2016- Id. 18445.