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Thyroid fine-needle aspiration (FNA) is a commonly used diagnostic cytological procedure in pediatric patients for the evaluation of thyroid nodules, triaging them for the detection of thyroid cancer. In recent years, greater attention has been paid to thyroid FNA in this setting, including the use of updated ultrasound score algorithms to improve accuracy and yield, especially considering the theoretically higher risk of malignancy of these lesions compared with the adult population, as well as to minimize patient discomfort. Moreover, molecular genetic testing for thyroid disease is an expanding field of research that could aid in distinguishing benign from cancerous nodules and assist in determining their clinical management. Finally, artificial intelligence tools can help in this task by performing a comprehensive analysis of all the obtained data. These advancements have led to greater reliance on FNA as a first-line diagnostic tool for pediatric thyroid disease. This review article provides an overview of these recent developments and their impact on the diagnosis and management of thyroid nodules in children.

Incidental thyroid carcinomas (ITCs) are a fairly frequent finding in daily routine practice, with papillary thyroid microcarcinoma being the most frequent entity. In our work, we isolated incidental cases arising in thyroids removed for other cytologically indeterminate and histologically benign nodules. We retrospectively retrieved cases with available thyroid Fine Needle Aspiration (FNA, 3270 cases), selecting those with an indeterminate cytological diagnosis (Bethesda classes III–IV, 652 cases). Subsequently, we restricted the analysis to surgically treated patients (163 cases) finding an incidental thyroid carcinoma in 22 of them. We found a 13.5% ITC rate, with ITCs representing 46.8% of all cancer histologically diagnosed in this indeterminate setting. Patients received a cytological diagnosis of Bethesda class III and IV in 41% and 59% of cases, respectively. All ITC cases turned out to be papillary thyroid microcarcinomas; 36% of cases were multifocal, with foci bilaterally detected in 50% of cases. We found an overall ITC rate concordant with the literature and with our previous findings. The assignment of an indeterminate category to FNA did not increase the risk of ITCs in our cohort. Rather, a strong statistical significance (p < 0.01) was found comparing the larger size of nodules that underwent FNA and the smaller size of their corresponding ITC nodule.

Fine needle aspiration (FNA) is the reference standard for the diagnosis of thyroid nodules. Matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI) has been successfully used to discriminate the proteomic profiles of benign and malignant thyroid FNAs within the scope of providing support to pathologists for the classification of morphologically borderline cases. However, real FNAs provide a limited amount of material due to sample collection restrictions. Ex vivo FNAs could represent a valuable alternative, increasing sample size and the power of statistical conclusions. In this study, we compared the real and ex vivo MALDI-MSI proteomic profiles, extracted from thyrocyte containing regions of interest, of 13 patients in order to verify their similarity. Statistical analysis demonstrated the mass spectra similarity of the proteomic profiles by performing intra-patient comparison, using statistical similarity systems. In conclusion, these results show that post-surgical FNAs represent a possible alternative source of material for MALDI-MSI proteomic investigations in instances where pre-surgical samples are unavailable or the number of cells is scarce.

In the field of thyroid neoplasms, the most interesting recent change regards the introduction of a new terminology for follicular-patterned thyroid tumors, named Noninvasive Thyroid Neoplasm with Papillary-like Nuclear Features (NIFTP). This pre-malignant tumor is considered to be the putative precursor of invasive carcinomas. However, given that several issues are still unresolved, the application of ancillary tools, based on omics-techniques, may improve the clinical management of these challenging cases. The present paper highlights the proteomic profiles of a series of NIFTPs submitted to Fine Needle Aspirations (FNAs) and analysed by MALDI-imaging in order to confirm the heterogeneous phenotype of nodules included in the present NIFTP terminology and to underline the necessity of more accurate biomarkers that can be used for their characterization. Ethical and economic implications in terms of healthcare costs, operative risks, morbidity, as well as the potential need for lifelong hormone replacement therapy, seem to be significant reasons to approach the characterization of NIFTPs using alternative tools such as MALDI-MSI.

Matrix-Assisted Laser Desorption/Ionization (MALDI)-Mass Spectrometry imaging (MSI) has been applied in various diseases aimed to biomarkers discovery. In this study diagnosis and prognosis of Hashimoto Thyroiditis (HT) in cytopathology by MALDI-MSI has been investigated. Specimens from a routine series of subjects who underwent UltraSound-guided thyroid Fine Needle Aspirations (FNAs) were used. The molecular classifier trained in a previous study was modified to include HT as a separate entity in the group of benign lesions, in the diagnostic proteomic triage of thyroid nodules. The statistical analysis confirmed the existence of signals that HT shares with hyperplastic lesions and others that are specific and characterize this subgroup. Statistically relevant HT-related peaks were included in the model. Then, the discriminatory capability of the classifier was tested in a second validation phase, showing a good agreement with cytological diagnoses. The possibility to overlap the molecular signatures of both the lymphocytes and epithelial cells components (ROIs or pixel-by-pixel analysis) confirmed the composite proteomic background of HT. These results open the way to their possible translation as alternative serum biomarkers of this autoimmune condition.

Introduction: Human herpes virus 8-related lymphoproliferative disorders are a complex and heterogeneous group of entities and some of them are eminently diagnosed by cytopathology. In a routine laboratory, these lesions account for less than 1% of the effusion fluids samples. However, they represent up to 30% of all the lymphoma diagnosis from effusion cytological samples and their consideration in the diagnostic flow chart is mandatory, especially in human immunodeficiency virus-positive patients. Methods: A retrospective series of cytological specimens from cavity effusions (n = 605) were analysed. Five human herpes virus 8-related lymphoproliferative processes were recruited. A combination of morphological criteria (enhanced with May-Grünwald Giemsa staining), cell block-based immunocytochemistry and flow cytometry were undertaken for final characterisation. Results: The identification of malignant cells may be difficult. Some specimens are particularly rich, easily leading to suspect a lymphoproliferative process, whereas in other cases, the presence of abundant reactive mesothelial cells, histiocytes, neutrophils, small reactive T and B lymphocytes may obscure the neoplastic process. The biological behaviour may be very heterogeneous and a standardised therapy for these cases is still lacking, although some patients may benefit from antiretroviral therapy in a human immunodeficiency virus setting. Conclusions: The present case series highlights some characteristic findings of these entities to reaffirm useful cytopathological diagnostic criteria, stressing the crucial role of the appropriate technical processing of effusion fluids to obtain the best performances.

In 2017, the WHO classification of tumours of the endocrine organs established the criteria for a NIFTP diagnosis. The present paper considers some aspects that are still debated or unresolved: the real incidence and clinical meaning of multifocal/multinodular lesions, the biological behaviour of micro-NIFTP, the sprinkling phenomenon and the corresponding modifications to the FNA reporting systems based on changes to the ROM. Moreover, the paper suggests possible scenarios for the clinical-pathological management of this entity. From the initial 1470 cases, a group of 68 NIFTPs was recruited in a 9 year-long period. The average age at diagnosis was 55 years. The average diameter of the lesion was 1.7 cm (0.1 cm–10 cm). In 41 cases (60.1%), the lesion was inserted in the context of a multinodular background. In 12 cases, the diagnosis was incidental and the pre- operative FNA was performed on a different target. In 10 out of 68 cases, there was a multifocal NIFTP; in 14.7% of patients, PTC-like nuclear features showed sprinkling phenomenon. The cytological revision allocated 21 cases (49%) to the SIAPEC TIR3 indeterminate class and a nuclear score 2 or 3 were identified in 25 smears. Multifocality is part of the spectrum of NIFTPs, that can arise in a multinodular background with variable sizes from microscopic lesions to very large ones. Cytopathological criteria such as an evaluation of the nuclear score may help the pathologists in promoting a NIFTP diagnosis in the preoperative setting.

The present study applies for the first time as Matrix-Assisted Laser Desorption/Ionization (MALDI) Mass Spectrometry Imaging (MSI) on real thyroid Fine Needle Aspirations (FNAs) to test its possible complementary role in routine cytology in the diagnosis of thyroid nodules. The primary aim is to evaluate the potential employment of MALDI-MSI in cytopathology, using challenging samples such as needle washes. Firstly, we designed a statistical model based on the analysis of Regions of Interest (ROIs), according to the morphological triage performed by the pathologist. Successively, the capability of the model to predict the classification of the FNAs was validated in a different group of patients on ROI and pixel-by-pixel approach. Results are very promising and highlight the possibility to introduce MALDI-MSI as a complementary tool for the diagnostic characterization of thyroid nodules.

Purpose: MALDI–MS imaging (MALDI–MSI) is an emerging technology that enables the spatial distribution of biomolecules within tissue to be combined with the traditional morphological information familiar to clinicians. Thus, for diagnostic or prognostic purposes, along with predicting response to therapeutic treatment, it is important to properly collect and handle biological specimens in order to avoid degradation or the formation of artifacts in the morphological structure and proteomic profile. Experimental design: In this work, the morphological and proteomic stability of thyroid fine needle aspiration biopsies in PreservCyt (up to 14 days) and CytoLyt (up to 7 days) solutions at 4 °C has been verified, by MALDI–MSI analysis. Moreover, a new measure has been introduced in order to assess the similarity of the obtained MALDI–MSI spectra, by equally taking into account the number of signals (fit and retrofit), and their intensities (Spearman's correlation and spectra overlap). Results: Results show no degradation of the cellular morphology and a good stability of the samples up to 14 days in PreservCyt solution. Conclusions and clinical relevance: Moreover, this protocol can be easily implemented in pathological units, allowing 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.

The main aim of the study was to assess the feasibility of matrix-assisted laser desorption/ionisation mass spectrometry imaging (MALDI-MSI) in the pathological investigation of Medullary Thyroid Carcinoma (MTC). Formalin-fixed paraffin-embedded (FFPE) samples from seven MTC patients were analysed by MALDI-MSI in order to detect proteomic alterations within tumour lesions and to define the molecular profiles of specific findings, such as amyloid deposition and C cell hyperplasia (CCH). nLC-ESI MS/MS was employed for the identification of amyloid components and to select alternative proteomic markers of MTC pathogenesis. Results highlighted the potential of MALDI-MSI to confirm the classic immunohistochemical methods employed for the diagnosis of MTC, with good sensitivity and specificity. Intratumoural amyloid components were also detected and identified, and were characterised by calcitonin, apolipoprotein E, apolipoprotein IV, and vitronectin. The tryptic peptide profiles representative of MTC and CCH were distinctly different, with four alternative markers for MTC being detected; K1C18, and three histones (H2A, H3C, and H4). Finally, a further 115 proteins were identified through the nLC-ESI-MS/MS analysis alone, with moesin, veriscan, and lumican being selected due to their potential involvement in MTC pathogenesis. This approach represents a complimentary strategy that could be employed to detect new proteomic markers of MTC. Statement of significance: Medullary thyroid carcinoma (MTC) is a rare endocrine malignancy that originates from the parafollicular C-cells of the thyroid. The diagnosis is typically established using a combination of fine-needle aspiration biopsy (FNAB) of a suspicious nodule along with the demonstrable elevation of serum biomarkers, such as calcitonin and carcinoembryonic antigen (CEA). Unfortunately, this combination is often associated with a high degree of false-positive results and this can lead to misdiagnosis and avoidable total th