Progress in Clinical Applications for the MALDI-Imaging Analysis of Thyroid Biopsies: Evaluation of Proteomic Stability in Preservative Solutions Isabella Piga (Presenter) University of Milano-Bicocca Presenter Bio: I obtained a bachelor degree in Chemistry from the University of Cagliari. The bachelor internship sparked an interest in analytical sciences and resulted in the following of the analytical chemistry master program at the University of Pisa. I decided to continue my academic career and I have attended the PhD School in Biochemistry and Molecular Biology at the University of Siena. As part of the PhD program, I have worked at the Center for Proteomics and Metabolomics in the Mass Spectrometry Imaging group, LUMC, the Netherlands. I am currently a Post-Doc at the University of Milano-Bicocca and my work is mostly focus on mass spectrometry imaging proteomics and clinical application. My main project is focused on the MALDI-Imaging analysis of thyroid fine needle aspiration biopsies and the classification of the thyroid nodule lesions.

Authors: Isabella Piga (1,2), Giulia Capitoli (3), Silvia Tettamanti (1), Vanna Denti (1), Andrew Smith (1), Clizia Chinello (1), Martina Stella (1), Davide Leni (4), Mattia Garancini (5), Stefania Galimberti (3), Fulvio Magni (1) and Fabio Pagni (2)
(1) University of Milano-Bicocca, Department of Medicine and Surgery, Clinical Proteomics and Metabolomics Unit, Italy (2) University of Milano-Bicocca, Department of Medicine and Surgery, Section of Pathology, Italy (3) University of Milano-Bicocca, Department of Medicine and Surgery, Centre of Biostatistics for Clinical Epidemiology, Italy (4) San Gerardo Hospital, Department of Radiology, Italy (5) San Gerardo Hospital, Department of Surgery, Italy

Thyroid lesions diagnosis is performed by image-guided fine needle aspiration biopsy (FNAB) regarded as a gold standard procedure. However, about 25% of FNABs are considered as “indeterminate for malignancy” and surgery is commonly recommended. Nevertheless, final histology highlight that 80% of them are benign lesions. Matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI) enables to explore the spatial distribution of biomarkers directly on cytological specimens, by integrating molecular and morphological evidence. This study focus on the proteomic profile stability of cytological samples in well-known preservative solutions, in order to have a standard methodology for the proteomic MALDI-MSI analysis of thyroid FNABs.

Introduction

Thyroid nodule lesion is one of the diseases diagnosed using liquid-based cytology. Thyroid FNABs are deposited into preservative solutions in order to lyse red blood cells, reduce the amount of mucus and maintain the morphological integrity during transportation and slide preparation. However, the proteomic stability of these liquid-based cytological 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 FNABs were collected from 14 patients (San Gerardo Hospital, Monza, Italy) and transferred into CytoLyt solution, centrifuged and re-suspended in PreservCyt solution. 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. 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. 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, 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.

Results

The washing steps in preservative solutions followed by cyto-deposition were able to guarantee high cellular adequacy. Cells morphology was generally satisfactory in different specimens from the same patient independently from the time of storage into the preservative media. With regard to conventional smear, our protocol has the advantage to be more efficient, reducing the sample-to-sample variability, and placing up to 8 spots into one ITO-slide. Moreover, the MALDI-MSI time of analysis of one cytospin-spot is drastically reduced (about 5h at 50x50µm raster width) compared to conventional smear (about 15h at 50x50µm raster width). 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 7 days. However longer storage time (2 months) in PreservCyt does not preserve the specimens, and the spectra overlap with t0 was only 50%. Moreover, the principal component analysis showed that the spectra profile from the same FNAB sample were clustered together independently from the time of storage in the preservative solution.

Conclusions & Discussion

This study represents a step forward towards the implementation of MALDI-MSI, combined with a trustworthy and robust methodology, into the cytopathology routine. This protocol allows 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. Moreover, we introduced a new mass spectra similarity score that equally take into account the number of signals (fit and retrofit) and their intensities (spearman’s correlation and spectra overlap).

FUNDING: This work was funded thanks to AIRC (Associazione Italiana per la Ricerca sul Cancro) MFAG GRANT 2016- Id. 18445.