## 2 Materials and Methods
### Data Preprocessing and Statistical Analyses  
The posttreatment of the obtained data was performed using MZmine2 version 2.39 ([[http://mzmine.github.io/](http://mzmine.github.io/)]([http://mzmine.github.io/](http://mzmine.github.io/)), accessed on 28 April 2019) [26]. Mass detection was performed using the mass detector tool. Chromatograms were generated with the ADAP chromatogram builder. Peaks were separated using the Peak extender module. Retention times were normalized with the retention time calibration module. Peaks were then aligned using the join aligner algorithm with a tolerance of 10 ppm in \(m/z\) and 1 min in retention time. The Human Metabolome Database version 5.0 (HMDB, [[http://www.hmdb.ca](http://www.hmdb.ca)]([http://www.hmdb.ca](http://www.hmdb.ca)), accessed on 7 January 2022) was used to predict ion identity with a mass tolerance of 15 ppm [27,28]. We only recorded ions predicted as [M + H]\({}^{+}\) adducts in the positive mode and [M - H]\({}^{-}\) adducts in the negative mode. The data were exported in csv format. We compiled the metabolites obtained from the positive and negative ionization modes into a made robust statistical analyses impossible. The samples from five patients were degraded and were therefore also excluded. The total cohort finally comprised 78 patients with TNs: 26 benign TNs (Bethesda II), 30 benign and 11 PTC cytologically indeterminate (Bethesda III-IV), and 11 PTC cytologically suspicious/malignant (Bethesda V-VI). The graphical representation of the experimental flowchart is shown in Figure 1. The demographic, biological, ultrasonographical, cytological, and histological characteristics of the patients and samples are summarized in Table 1, and fully described in Supplementary Tables S1-S4. Briefly, the cohort included 74.4% women, aged 52.6 years (\(\pm\)15.8), mainly euthyroidic (83.3%), with a TN of diameter 23.1 mm (\(\pm\)10.4). Patients with benign TNs presented an adenoma or a non-neoplastic nodule in 50% of the cases. Among the patients with a PTC, 54.5% of the cases were of a classical variant. \begin{table}
\begin{tabular}{l c c c c} \hline \hline \multirow{2}{*}{Characteristics} & & **All** & **Benign** & **Malignant** \\  & & **\% (nb)** & **\% (nb)** & **\% (nb)** \\ \cline{3-5}  & & **78** & **56** & **22** \\ \hline Gender & F & 74.4 (58) & 76.8 (43) & 68.2 (15) \\ Age (mean \(\pm\)SD) & & 52.6 (\(\pm\)15.8) & 56.1 (\(\pm\)14.6) & 43.7 (\(\pm\)15.4) \\ Hashimoto & & 11.4 (8) & 6.3 (3) & 22.7 (5) \\ Thyroid function & Euthyroidism & 83.3 (65) & 82.1 (46) & 86.4 (19) \\  & Hypothyroidism & 7.7 (6) & 7.1 (4) & 9.1 (2) \\  & Hyperthyroidism & 7.7 (6) & 8.9 (5) & 4.5 (1) \\ \hline EU-TIRADS & 3 & 23.1 (18) & 32.1 (18) & 0 \\  & 4 & 46.2 (36) & 48.2 (27) & 36.4 (8) \\  & 5 & 29.5 (23) & 17.9 (10) & 59.1 (13) \\ US size (mm, mean \(\pm\)SD) & & 23.1 (\(\pm\)10.4) & 25.4 (\(\pm\)10.2) & 17.4 (\(\pm\)8.9) \\ \hline \multirow{2}{*}{Cytrologic classification (Bethesda)} & II & 33.3 (26) & 46.4 (26) & 0 \\  & III & 20.5 (16) & 17.9 (10) & 27.3 (6) \\ \cline{1-1}  & IV & 32.1 (25) & 35.7 (20) & 22.7 (5) \\ \cline{1-1}  & V & 6.4 (5) & 0 & 22.7 (5) \\ \cline{1-1}  & VI & 7.7 (6) & 0 & 27.3 (6) \\ \hline \hline \end{tabular}
\end{table} 
Table 1: Demographic characteristics of the cohort of patients. Figure 1: Graphical representation of the flowchart of the study. Patients with thyroid nodules (TNs) underwent ultrasound-guided fine-needle aspiration cytology (FNAC) according to the recommendations of the EU-TIRADS classification [24]. Patients were managed in accordance with the Bethesda system for reporting thyroid cytopathology (TBSRTC) and the ATA recommendations [13; 14]. For Bethesda 2 lesions (i.e., benign lesions), no surgery was performed, except for compressive nodules. For other cytology results, only patients who underwent surgery with a histological diagnosis of benign or malignant lesion were included.