Genetic Predisposition to Familial Nonmedullary Thyroid Cancer: An Update of Molecular Findings and State-of-the-Art Studies

Familial thyroid cancer has become a well-recognized entity in patients with thyroid cancer originating from follicular cells, that is, nonmedullary thyroid carcinoma. The diagnosis of familial thyroid cancer provides an opportunity for early detection and possible prevention in family members. Understanding the syndromes associated with familial thyroid cancer allows clinicians to evaluate and treat patients for coexisting pathologic conditions. About five percents of patients with well-differentiated thyroid carcinoma have a familial disease. Patients with familial non-medullalry thyroid cancer have more aggressive tumors with increased rates of extrathyroid extension, lymph node metastases, and frequently show the phenomenon of “anticipation” (earlier age at disease onset and increased severity in successive generations). So far, four predisposition loci have been identified in relatively rare extended pedigrees, and association studies have identified multiple predisposing variants for differentiated thyroid cancer. This suggests that there is a high degree of genetic heterogeneity and that the development of this type of tumor is a multifactorial and complex process in which predisposing genetic variants interact with a number of incompletely understood environmental risk factors. Thus, the search for the causative variants is still open and will surely benefit from the new technological approaches that have been developed in recent years

What Is New in Thyroid Cancer: The Special Issue of the Journal Cancers

The incidence of thyroid cancer has increased over the past 3 to 4 decades. Nonetheless, the mortality from thyroid cancer has remained stable. The thyroid gland may develop nodules encompassing several types of cell proliferation, from frankly benign to very aggressive forms with many intermediate challenging variants. For this reason, there is growing interest in evaluating thyroid nodules from many points of view, from the clinical to the molecular aspects, in the search for innovative diagnostic and prognostic parameters. The aim of this Special Issue was to provide an overview of recent developments in understanding the biology and molecular oncology of thyroid tumors of follicular cell derivation and their repercussions on the diagnosis, prognosis, and therapy. The contributions of many experts in the field made up a Special Issue of Cancers journal, that focusing on different aspects, including mechanistic and functional facets, gives the status of art of clinical and biological perspectives of thyroid cancer

Molecular diagnosis of carcinomas of the thyroid gland.

Our understanding of the molecular pathology of thyroid cancer has progressed significantly. It is now apparent that thyroid tumors show a very good correlation between genotype and phenotype, a correlation that is much stronger than that observed in tumors of many other organs. Activation of classic oncogenes (BRAF, RAS, RET) activate MAPK signalling. Other pathways like the PI3K/PTEN/AKT cascade are also active in many thyroid tumors. The analysis of molecular profiles is generating data that can be applied to improve patient management. The common occurrence of thyroid nodules in the general population and the widespread use of fine needle aspiration for the preoperative diagnosis of thyroid nodules creates an unprecedented opportunity to apply what we have learnt from the molecular alterations of thyroid cancer to the clinical arena

Allele Specific Locked Nucleic Acid Quantitative PCR (ASLNAqPCR): An Accurate and Cost-Effective Assay to Diagnose and Quantify KRAS and BRAF Mutation

The use of tyrosine kinase inhibitors (TKIs) requires the testing for hot spot mutations of the molecular effectors downstream the membrane-bound tyrosine kinases since their wild type status is expected for response to TKI therapy. We report a novel assay that we have called Allele Specific Locked Nucleic Acid quantitative PCR (ASLNAqPCR). The assay uses LNA-modified allele specific primers and LNA-modified beacon probes to increase sensitivity, specificity and to accurately quantify mutations. We designed primers specific for codon 12/13 KRAS mutations and BRAF V600E, and validated the assay with 300 routine samples from a variety of sources, including cytology specimens. All were analyzed by ASLNAqPCR and Sanger sequencing. Discordant cases were pyrosequenced. ASLNAqPCR correctly identified BRAF and KRAS mutations in all discordant cases and all had a mutated/wild type DNA ratio below the analytical sensitivity of the Sanger method. ASLNAqPCR was 100% specific with greater accuracy, positive and negative predictive values compared with Sanger sequencing. The analytical sensitivity of ASLNAqPCR is 0.1%, allowing quantification of mutated DNA in small neoplastic cell clones. ASLNAqPCR can be performed in any laboratory with real-time PCR equipment, is very cost-effective and can easily be adapted to detect hot spot mutations in other oncogenes

RET mutation and increased angiogenesis in medullary thyroid carcinomas

Advanced medullary thyroid cancers (MTCs) are now being treated with drugs that inhibit receptor tyrosine kinases, many of which involved in angiogenesis. Response rates vary widely, and toxic effects are common, so treatment should be reserved for MTCs likely to be responsive to these drugs. RET mutations are common in MTCs, but it is unclear how they influence the microvascularization of these tumors. We examined 45 MTCs with germ-line or somatic RET mutations (RETmut group) and 34 with wild-type RET (RETwt). Taqman Low-Density Arrays were used to assess proangiogenic gene expression. Immunohistochemistry was used to assess intratumoral, peritumoral and nontumoral expression levels of VEGFR1, R2, R3, PDGFRa, PDGFB and NOTCH3. We also assessed microvessel density (MVD) and lymphatic vessel density (LVD) based on CD31-positive and podoplanin-positive vessel counts, respectively, and vascular pericyte density based on staining for a-smooth muscle actin (a-SMA), a pericyte marker. Compared with RETwt tumors, RETmut tumors exhibited upregulated expression of proangiogenic genes (mRNA and protein), especially VEGFR1, PDGFB and NOTCH3. MVDs and LVDs were similar in the two groups. However, microvessels in RETmut tumors were more likely to be a-SMA positive, indicating enhanced coverage by pericytes, which play key roles in vessel sprouting, maturation and stabilization. These data suggest that angiogenesis in RETmut MTCs may be more intense and complete than that found in RETwt tumors, a feature that might increase their susceptibility to antiangiogenic therapy. Given their increased vascular pericyte density, RETmut MTCs might also benefit from combined or preliminary treatment with PDGF inhibitors

TERT Promoter Mutations in Papillary Thyroid Microcarcinomas

Small papillary thyroid carcinomas have contributed to the worldwide increased incidence of differentiated thyroid cancer observed over the past decades. However, the mortality rate has not changed over the same period of time, raising questions about the possibility that thyroid cancer patients, especially those with small tumors, are overdiagnosed and overtreated. Molecular prognostic marker able to discriminate aggressive thyroid cancers from those with an indolent course would be of great relevance to tailor the therapeutic approach and reduce overtreatment. Mutations in the TERT promoter were recently reported to correlate strongly with aggressiveness in advanced forms of thyroid cancer, holding promise for a possible clinical application. The occurrence and potential clinical relevance of TERT mutations in papillary thyroid microcarcinomas (mPTCs) is currently unknown. This study aimed to analyze the occurrence of two TERT promoter mutations (-124C>T and -146C>T) and their potential association with unfavorable clinical features in a large cohort of mPTCs

Anoctamin 1 is Apically Expressed on Thyroid Follicular Cells and Contributes to ATP- and Calcium-Activated Iodide Efflux

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Increased expression of pro-angiogenic factors and vascularization in thyroid hyperfunctioning adenomas with and without TSH receptor activating mutations

Autonomously functioning thyroid nodules (AFTN) are known to receive an increased blood influx necessary to sustain their high rate of growth and hormone production. Here, we investigated the expression of hematic and lymphatic vases in a series of 20 AFTN compared with the contralateral non-tumor tissues of the same patients, and the transcript levels of proteins involved in the control of vascular proliferation, including the vascular endothelial growth factor (VEGF) and platelet-derived growth factors (PDGF) and their receptors and the endothelial nitric oxide synthase (eNOS). In parallel, the expression of the differentiation markers sodium/iodide symporter (NIS), thyroperoxidase (TPO), thyroglobulin (Tg), and TSH receptor (TSHR) was also investigated. The data were further analyzed comparing subgroups of tumors with or without mutations in the TSHR gene. Analysis by means of CD31 and D2-40 immunostaining showed in AFTN an increased number of hematic, but not lymphatic, vessels in parallel with an enhanced proliferation rate shown by increased Ki67 staining. Quantitative RT-PCR analysis revealed an increase of VEGF, VEGFR1 and 2, PDGF-A, PDGF-B, and eNOS expression in tumor versus normal tissues. Also, higher transcript levels of NIS, TPO, and Tg were detected. Comparison of the two subgroups of samples revealed only few differences in the expression of the genes examined. In conclusion, these data demonstrate an increased expression of angiogenesis-related factors associated with an enhanced proliferation of hematic, but not lymphatic, vessels in AFTNs. In this context, the presence of TSHR mutations may only slightly influence the expression of pro-angiogenic growth factors

Different Methods in HPV Genotyping of Anogenital and Oropharyngeal Lesions: Comparison between VisionArray® Technology, Next Generation Sequencing, and Hybrid Capture Assay

(1) Background: Human papillomaviruses (HPVs) are known to be related to the development of about 5% of all human cancers. The clinical relevance of HPV infection has been deeply investigated in carcinomas of the oropharyngeal area, uterine cervix, and anogenital area. To date, several different methods have been used for detecting HPV infection. The aim of the present study was to compare three different methods for the diagnosis of the presence of the HPV genome. (2) Methods: A total of 50 samples were analyzed. Twenty-five of them were tested using both next generation sequencing (NGS) and VisionArray® technology, the other 25 were tested using Hybrid Capture (HC) II assay and VisionArray® technology. (3) Results: A substantial agreement was obtained using NGS and VisionArray® (κ = 0.802), as well as between HC II and VisionArray® (κ = 0.606). In both analyses, the concordance increased if only high risk HPVs I(HR-HPVs) were considered as “positive”. (4) Conclusions: Our data highlighted the importance of technical choice in HPV characterization, which should be guided by the clinical aims, costs, starting material, and turnaround time for results

Papillary thyroid carcinoma tall cell variant shares accumulation of mitochondria, mitochondrial DNA mutations, and loss of oxidative phosphorylation complex I integrity with oncocytic tumors

open12siMDL was supported by the Associazione Italiana per la Ricerca sul Cancro (AIRC) ‘Bruna Martelli’ Fellowship.Papillary thyroid carcinoma tall cell variant (PTC-TCV), a form of PTC regarded as an aggressive subtype, shares several morphologic features with oncocytic tumors. Pathogenic homoplasmic/highly heteroplasmic somatic mitochondrial DNA (mtDNA) mutations, usually affecting oxidative phosphorylation (OXPHOS) complex I subunits, are hallmarks of oncocytic cells. To clarify the relationship between PTC-TCV and oncocytic thyroid tumors, 17 PTC-TCV and 16 PTC non-TCV controls (cPTC) were subjected to: (1) transmission electron microscopy (TEM) to assess mitochondria accumulation, (2) next-generation sequencing to analyze mtDNA and nuclear genes frequently mutated in thyroid carcinoma, and (3) immunohistochemistry (IHC) for prohibitin and complex I subunit NDUFS4 to evaluate OXPHOS integrity. TEM showed replacement of cytoplasm by mitochondria in PTC-TCV but not in cPTC cells. All 17 PTC-TCV had at least one mtDNA mutation, totaling 21 mutations; 3/16 cPTC (19%) had mtDNA mutations (p < 0.001). PTC-TCV mtDNA mutations were homoplasmic/highly heteroplasmic, 16/21 (76%) mapping within mtDNA-encoded complex I subunits. MtDNA mutations in cPTC were homoplasmic in 2 cases and at low heteroplasmy in the third case, 2/3 mapping to mtDNA-encoded complex I subunits; 2/3 cPTC with mtDNA mutations had small tall cell subpopulations. PTC-TCV showed strong prohibitin expression and cPTC low-level expression, consistent with massive and limited mitochondrial content, respectively. All 17 PTC-TCV showed NDUFS4 loss (partial or complete) and 3 of 16 cPTC (19%) had (partial) NDUFS4 loss, consistent with lack of complex I integrity in PTC-TCV (p < 0.001). IHC loss of NDUFS4 was associated with mtDNA mutations (p < 0.001). Four BRAF V600E mutated PTCs had loss of NDUSF4 expression limited to neoplastic cell subpopulations with tall cell features, indicating that PTCs first acquire BRAF V600E and then mtDNA mutations. Similar to oncocytic thyroid tumors, PTC-TCV is characterized by mtDNA mutations, massive accumulation of mitochondria, and loss of OXPHOS integrity. IHC loss of NDUFS-4 can be used as a surrogate marker for OXPHOS disruption and to reliably diagnose PTC-TCV.openTsybrovskyy, Oleksiy; De Luise, Monica; Biase, Dario; Caporali, Leonardo; Fiorini, Claudio; Gasparre, Giuseppe; Carelli, Valerio; Hackl, Dominik; Imamovic, Larisa; Haim, Silke; Sobrinho‐Simões, Manuel; Tallini, GiovanniTsybrovskyy, Oleksiy; De Luise, Monica; Biase, Dario; Caporali, Leonardo; Fiorini, Claudio; Gasparre, Giuseppe; Carelli, Valerio; Hackl, Dominik; Imamovic, Larisa; Haim, Silke; Sobrinho‐Simões, Manuel; Tallini, Giovann