Metabolic biomarkers of neoplastic cells in non-medullary differentiated thyroid carcinoma

Although thyroid nodules are common, only 10-15% are malignant and require surgical treatment. A systematic approach to their evaluation is important to avoid unnecessary surgery. Fine-needle aspiration biopsy (FNAB) has substantially improved the diagnostic accuracy. However, despite FNAB effectiveness, about 10% of all specimens are classified as “indeterminate”. In those cases, clinicians usually recommend surgical excision for a definitive diagnosis. Because only 15% to 20% of these lesions are malignant, up to 85% of the patients in this subgroup may undergo unnecessary surgery. In this paper, we want to develop an HR-MAS NMR based-approach presuming that metabolic variations will pre-empt the development of morphologic modifications associated with malignancy. Analyzing a series of 72 thyroid lesions (28 papillary thyroid carcinomas, 40 “indeterminate” lesions, and 4 benign nodules), we want to show that the HR-MAS technique is suitable not only to highlight metabolic differences between normal and tumoral thyroid tissues, but can also discriminate malignant from benign tumoral tissues. The principal aim is to transfer this technique to FNABs, in order to answer to diagnostic problems encountered by clinicians and improve the clinical management of patients with a “indeterminate” cytological diagnosis

DICER1 somatic mutations strongly impair miRNA processing even in benign thyroid lesions

The alteration of miRNA processing is a driver event in several tumors including thyroid cancer. In particular, somatic DICER1 mutations, reported in follicular-patterned lesions, are shared by benign as well as malignant tumors. In the present study, we investigated the effects of alterations in the miRNA processing genes on the miRNA profile. The study included 19 follicular adenomas (FAs) and 22 follicular variant of papillary thyroid carcinomas (FVPTCs). The mutational status in the hot spot regions of DICER1, DROSHA, TARBP2, DGCR8 and the most commonly affected genes in thyroid tumors was investigated on both tumor and paired normal tissues. The miRNA profile and the mRNA expression levels of DICER1, DROSHA, TARBP2, DGCR8 and XPO5 were also evaluated. Two DICER1 RNase IIIb domain mutations were found in FAs. These lesions presented a considerable loss of 5p miRNAs. Fifteen miRNAs were specifically deregulated in DICER1-mutant lesions compared to FAs and FVPTCs. These miRNAs regulate crucial pathways in cancer such as Hippo, p53 and TGF-beta signalling. DICER1 somatic mutations in the RNase IIIb domain are not specific for malignancy, but the miRNA imbalance that they cause is remarkable, especially with regard to the loss of 5p miRNAs. DICER1-mutant lesions have a characteristic miRNA deregulation, which is different from that of FVPTCs; nevertheless, this impairment is consistent with malignant transformation. Further studies providing the real risk of malignancy associated with DICER1 mutations and the evolution of DICER1-mutant lesions are needed to make them useful in the clinical practice

'Incidental' and 'non-incidental' thyroid papillary microcarcinomas are two different entities

Papillary thyroid microcarcinomas (microPTC) may be 'incidental' (Inc-microPTC), occasionally found at histology after surgery for benign disease or 'non-incidental' (Non-Inc-microPTC), diagnosed on clinical grounds. It is unclear whether these different microPTC reflect the same disease. The aim of the study was to compare Inc-microPTC and Non-Inc-microPTC for clinical and histological features as well as for serum TSH, a known factor involved in PTC development

RET mutated C-cells proliferate more rapidly than non-mutated neoplastic cells

A statistically significant higher prevalence of the RET p.Met918Thr somatic mutation, identified by direct sequencing, was previously reported in MTC >2 cm than in smaller tumors. Aim of this study was to correlate the full RET and RAS mutation profile, identified by a Next Generation Sequencing approach, with the growth rate, proliferation and tumor size of MTC. Data of 149 sporadic MTC patients were correlated with RET mutations and Ki67 positivity. Eighty-one cases had a somatic RET mutation, 40 a RAS mutation and 28 were negative. A statistically significant higher prevalence of RET mutations was found in MTC >2 cm. A higher prevalence of RET more aggressive mutations, higher allelic frequencies and, higher percentage of Ki67 positive cells were found in larger tumors which had also a worse outcome. Our study highlights the predominant role of RET somatic mutations in MTC tumorigenesis. We demonstrate that RET mutation prevalence and allelic frequency (AF) are significantly higher in larger tumors. Based on these results, we can conclude that RET mutated C-cells's growth and proliferation are more rapid than those of non-mutated cells and give origin to bigger and more aggressive MTC

mRNA and miRNA expression profiling of follicular variant of papillary thyroid carcinoma with and without distant metastases

Follicular Variant of Papillary Thyroid Carcinoma (FVPTC) is usually associated with a good outcome. Nevertheless, in rare cases, it develops distant metastases (1–9%). Our goal was to investigate whether mRNA and miRNA expression profiles may help distinguish between metastatic versus non-metastatic FVPTCs. Twenty-four primary FVPTCs, 12 metastatic and 12 non-metastatic, with similar clinicopathological features were selected and analyzed by nanoString nCounter technology using two distinct panels for expression analysis of 740 mRNA and 798 miRNAs. Data analysis was performed using the nanoString nSolver 3.0 software. Forty-seven mRNA and 35 miRNAs were differentially expressed between the two groups. Using these mRNA and miRNAs, metastatic and non-metastatic FVPTCs were clearly divided into two distinct clusters. Our results indicate that FVPTCs with metastatic abilities have different expression profiles compared to the non-metastatic. A prospective validation is needed to evaluate the usefulness of this molecular approach in the early identification of high-risk FVPTCs

Functional characterization of a CDKN1B mutation in a Sardinian kindred with multiple endocrine neoplasia type 4 (MEN4)

Inactivating germline mutations of the CDKN1B gene, encoding for the nuclear cyclin-dependent kinase inhibitor p27kip1 protein, have been reported in patients with multiple endocrine neoplasia type 4 (MEN4), a MEN1-like phenotype without MEN1 mutations. The aim of this study was to in vitro characterize the germline CDKN1B mutation c.374_375delCT (S125X) we detected in a patient with MEN4. The proband was affected by multiglandular primary hyperparathyroidism and gastro-entero-pancreatic tumors. We carried out subcellular localization experiments transfecting into eukaryotic HeLa and GH3 cell lines plasmid vectors expressing the CDKN1B wild type (wt) or mutant cDNA. Western blot studies showed that fusion proteins were expressed at equal levels. The mutated protein was shorter compared to the wt protein and lacked the highly conserved C-terminal domain, which includes the bipartite nuclear localization signal at amino acids 152/153 and 166/168. In HeLa and GH3 cells wt p27 localized in the nucleus whereas the p27_S125X protein was retained in the cytoplasm predicting the loss of tumor suppressive function. The proband's tumoral parathyroid tissue did not show allelic loss, since wt and mutant alleles were both present by sequencing the somatic DNA. Immunohistochemistry showed a complete loss of nuclear p27 expression in the parathyroid adenoma removed by the patient at the second surgery. In conclusion, our study confirms the pathogenic role of the c.374_375delCT CDKN1B germline mutation in a patient with MEN4

Evaluation of formalin-fixed paraffin-embedded tissues in the proteomic analysis of parathyroid glands

<p>Abstract</p> <p>Background</p> <p>Proteomic research in the field of parathyroid tissues is limited by the very small dimension of the glands and by the low incidence of cancer lesions (1%). Formalin-fixed paraffin-embedded (FFPE) tissue specimens are a potentially valuable resource for discovering protein cancer biomarkers. In this study we have verified the applicability of a heat induced protein extraction from FFPE parathyroid adenoma tissues followed by a gel-based or gel-free proteomic approach in order to achieve protein separation and identification.</p> <p>Results</p> <p>The best results for high quality MS spectra and parameters, were obtained by using a gel-free approach, and up to 163 unique proteins were identified. Similar results were obtained by applying both SDS-out and SDS-out + TCA/Acetone techniques during the gel-free method. Western blot analysis carried out with specific antibodies suggested that the antigenicity was not always preserved, while specific immunoreactions were detected for calmodulin, B box and SPRY domain-containing protein (BSPRY), peroxiredoxin 6 (PRDX 6) and parvalbumin.</p> <p>Conclusions</p> <p>In spite of some limitations mainly due to the extensive formalin-induced covalent cross-linking, our results essentially suggest the applicability of a proteomic approach to FFPE parathyroid specimens. From our point of view, FFPE extracts might be an alternative source, especially in the validation phase of protein biomarkers when a large cohort of samples is required and the low availability of frozen tissues might be constraining.</p

Low elasticity of thyroid nodules at ultrasound elastography is correlated with malignancy, degree of fibrosis and high expression of galectin-3 and fibronectin-1

ackground: Thyroid ultrasound (US) elastography provides an estimation of tissue stiffness and is helpful to differentiate malignant from benign lesions. Tissue proprieties and molecules causing stiffness are not established. The aim of the study was to correlate US elastography findings with tissue properties in thyroid nodules. Methods: A total of 115 thyroid nodules from 112 patients who underwent surgery for the presence of Thy 3 (indeterminate) cytology (n = 67), Thy 4-5 (suspicious - indicative of carcinoma) cytology (n = 47), or large goiter in the presence of Thy 2 cytology (n = 1) and suspicious US features were examined by US elastography. Tissues obtained after surgery were characterized for cell number, microvessel density, fibrosis, and expression of galectin-3 (Gal-3) and fibronectin-1 (FN-1). Results: Low elasticity on qualitative US elastography (LoEl) was found in 66 nodules (one benign and 65 carcinomas); high elasticity (HiEl) was found in 49 nodules (46 benign and three carcinomas; p &lt; 0.0001). Quantitative analysis, performed in 24 nodules and expressed as elastic ratio between the strain of the nodule and that of the surrounding thyroid parenchyma, showed a mean of 1.90 (interquartile range [IQR] 1.18-2.77) in 14 nodules with LoEl, and a mean of 1.01 (IQR 0.91-1.10) in 10 nodules with HiEl (p = 0.002). Stiffness did not correlate with cell number and was inversely correlated with microvessel density. Fibrosis was higher in nodules with LoEl than in those with HiEl (p = 0.009) and in carcinomas than in benign nodules (p = 0.02). Fibrosis was higher in nodules with high expression of Gal-3 (p &lt; 0.001) and FN-1 (p = 0.004). Fibrosis and expression of Gal-3 and FN-1 were higher in the classic compared with the follicular variant of papillary thyroid carcinoma and lower in follicular adenomas. Conclusions: Low elasticity at US elastography is highly correlated with malignancy. Nodule stiffness is correlated with fibrosis and expression of Gal-3 and FN-1. These features are more evident in the classic than in the follicular variant of papillary thyroid carcinoma

Firm mass in thyroid of an elderly patient: not always cancer

Summary In elderly patients presenting with a solid thyroid mass, the differential diagnosis between benign and malignant lesion is not always straightforward. We present the case of an 85-year-old woman with fever and an enlarged, firm and painful thyroid mass. Blood exams documented a mild thyrotoxicosis with a moderate inflammatory status. Thyroid scintiscan showed an absent uptake of 131I. Ultrasound and CT scan documented a 3 cm hypoechoic nodule with infiltration of the sternocleidomastoid muscle, very suspicious for neoplastic nature. Fine-needle aspiration and tru-cut biopsy were performed. During biopsy, the lesion was partially drained and a brownish fluid was extracted. The culture resulted positive for Klebsiella pneumoniae whereas the pathological analysis of the specimen was not conclusive due to the presence of an intense inflammatory response. A targeted oral antibiotic therapy was then initiated, obtaining only a partial response thus, in order to achieve a definite diagnosis, a minimally invasive hemithyroidectomy was performed. The pathological analysis documented acute suppurative thyroiditis and the clinical conditions of the patient significantly improved after surgical removal of thyroid abscess. In elderly patients with a solid thyroid mass, although neoplastic origin is quite frequent, acute suppurative thyroiditis should be considered as a differential diagnosis. Learning points: A solid and rapidly growing thyroid mass in elderly patients can hide a multifaceted variety of diseases, both benign and malign. A multidisciplinary team (endocrinologist, surgeon, radiologist and pathologist) could be necessary in order to perform a correct differential diagnosis and therapeutic approach. Surgery can be decisive not only to clarify a clinically uncertain diagnosis, but also to rapidly improve the clinical conditions of the patient

Proteomic approach used in the diagnosis of Riedel's thyroiditis: a case report

<p>Abstract</p> <p>Introduction</p> <p>Riedel's thyroiditis, a rare thyroid disease, can be difficult to diagnose prior to surgical removal and can be confused with malignancy both clinically and cytologically.</p> <p>Case presentation</p> <p>We report the case of a 72-year-old Caucasian woman who presented with a goiter, which showed a rapid increase in size at ultrasound check, suggesting malignancy. Because of inconclusive cytology, a total thyroidectomy was performed. Fine-needle aspiration of the removed thyroid was processed by two-dimensional electrophoresis, and the proteome was compared with both anaplastic cancer and control samples. Significant differentially expressed protein spots were identified by Western blot analysis by using specific antibodies.</p> <p>Conclusions</p> <p>The protein pattern of Riedel's fine-needle aspiration revealed a superimposition with that of the control samples. The comparison of the protein pattern of Riedel's thyroiditis fine-needle aspiration with that of anaplastic cancer showed evidence of a different expression of ferritin heavy chains, ferritin light chains, and haptoglobins, as previously reported in thyroid cancers. Therefore, we performed Western blot analysis of these proteins and validated that their expression levels were low or absent in Riedel's thyroiditis and control samples despite the high concentrations present in fine-needle aspiration anaplastic samples. The concurrent absent or low expression levels of haptoglobin, ferritin light chain, and ferritin heavy chain in Riedel's thyroiditis fine-needle aspiration samples strongly indicate the benign nature of the thyroid lesion. These results suggest the potential applicability of fine-needle aspiration proteome analysis for Riedel's thyroiditis diagnosis.</p