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 < 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 < 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

ALK rearrangement in a large series of consecutive non-small cell lung cancers: Comparison between a new immunohistochemical approach and fluorescence in situ hybridization for the screening of patients eligible for crizotinib treatment

Context. - Echinoderm microtubule associated protein-like 4-anaplastic lymphoma receptor tyrosine kinase (EML4-ALK) translocation has been described in a subset of patients with non-small cell lung cancer (NSCLC) and has been shown to have oncogenic activity. Fluorescence in situ hybridization (FISH) is used to detect ALK-positive NSCLC, but it is expensive, time-consuming, and difficult for routine application. Objective. - To evaluate the potential role of immunohistochemistry (IHC) as a screening tool to identify candidate cases for FISH analysis and for ALK inhibitor therapy in NSCLC. Design. - We performed FISH and IHC for ALK and mutational analysis for epidermal growth factor receptor (EGFR) and KRAS in 523 NSCLC specimens. We conducted IHC analysis with the monoclonal antibody D5F3 (Ventana Medical Systems, Tucson, Arizona) and a highly sensitive detection system. We also performed a MassARRAY-based analysis (Sequenom, San Diego, California) in a small subset of 11 samples to detect EML4-ALK rearrangement. Results. - Of the 523 NSCLC specimens, 20 (3.8%) were positive for ALK rearrangement by FISH analysis. EGFR and KRAS mutations were identified in 70 (13.4%) and 124 (23.7%) of the 523 tumor samples, respectively. ALK rearrangement and EGFR and KRAS mutations were mutually exclusive. Of 523 tumor samples analyzed, 18 (3.4%) were ALK+ by IHC, 18 samples (3.4%) had concordant IHC and FISH results, and 2 ALK+ cases (0.3%) by FISH failed to show ALK protein expression. In the 2 discrepant cases, we did not detect any mass peaks for the EML4-ALK variants by MassARRAY. Conclusions. - Our results show that IHC may be a useful technique for selecting NSCLC cases to undergo ALK FISH analysis

miRNA expression profiling of Noninvasive follicular thyroid neoplasm with papillary-like nuclear features: more light on its behavior

Follicular variant of PTC (FVPTC) is a variant of well differentiated papillary thyroid carcinoma. FVPTCs comprise encapsulated and infiltrative forms with different histo-logical features and clinical behavior, but according cytomorphological features the forms with indolent behavior are not significant different from those with aggressive phenotype. Since the encapsulated forms with no capsular invasion show an indolent clinical behavior similar to benign lesion, recently , an international panel of patholo-gists and clinicians has reclassified these lesions as a distinct class of thyroid tumors termed as “Non-invasive follicular thyroid neoplasms with papillary-like nuclear fea-tures” (NIFTP). To better classify these tumors by a molecular viewpoint and to evaluate new diagno-stic tool to improve pre-surgical differential diagnosis between NIFTPs, Follicular Adenomas (FAs) and Infiltrative FVPTCs, an expression profiling of 798 miRNAs was performed. miRNA expression levels were detected in 54 thyroid tumors (18 Follicular adeno-mas, 19 NIFTPs and 17 Infiltrative FVPTCs) using an highly reproducible and sensi-tive method, nCounter Nanostring assay. A set of differentially expressed miRNAs was identified. miR-146-5p, miR-221-5p, miR-222-3p, miR-30e-3p and miR-152-3p were able to discriminate benign from ma-lignant lesions with a very high level of significance (p-value<0.001); miR-146-5p, miR-199a-5p, miR-199b-5p, miR-1915-3p and miR-148b-3p were most probably as-sociated with a infiltrative growth of FVPTCs. miR-152-3p, miR-185-5p, miR-574-3p were found to be strongly down-regulated in NIFTPs compared with FAs whereas a significant overexpression of miR-10a-5p was reported in NIFTPs compared with infiltrative forms of FVPTC. In conclusion, our data suggest that a panel of these markers could have a high diag-nostic potential and could be applied in the preoperative assessment of thyroid nodu-les, mainly for the lesions that cannot be reliably distinguished on cytology alone and are classified as indeterminate nodules

CARATTERIZZAZIONE MOLECOLARE DI UNA SERIE DI CARCINOMI TIROIDEI POCO DIFFERENZIATI E DELLE RELATIVE METASTASI LINFONODALI

I carcinomi tiroidei sono le neoplasie maligne più frequenti del sistema endocrino e vengono di norma classificati in ben differenziati (papillari e follicolari), poco differenziati (PDC) e indifferenziati o anaplastici in base al grado di differenziazione cellulare. I PDC occupano una posizione intermedia dal punto di vista morfologico, patogenetico e come comportamento clinico tra i carcinomi ben differenziati e gli indifferenziati; infatti, pur mantenendo un certo grado di differenziazione cellulare, la loro capacità di metastatizzare e di infiltrare i tessuti paratiroidei è più vicina a quella degli anaplastici e hanno quindi una prognosi sicuramente peggiore rispetto alle lesioni neoplastiche ben differenziate. Inoltre, dal punto di vista genetico, possono presentare mutazioni (BRAF e RAS) caratteristiche che rappresentano un evento precoce nella tumorigenesi, ma anche mutazioni a carico di geni (ad es. TP53) coinvolti nella ulteriore progressione neoplastica, frequentemente riscontrati negli anaplastici. Le caratteristiche prime descritte hanno fatto supporre che alcune forme di lesioni poco differenziate deriverebbero da un carcinoma pre-esistente ben differenziato, in seguito ad una serie successiva di alterazioni che determinano il fenotipo più maligno. Mentre si attribuisce un’origine diversa a quei PDC che non presentano lo stesso pattern genetico e sono privi di aree residue ben differenziate. I PDC rappresentano, quindi, un gruppo eterogeneo di tumori, di difficile approccio clinico-patologico; pertanto sono uno degli argomenti oggetto di maggiore dibattito nell’ambito della patologia tiroidea. Lo studio di questi carcinomi, attraverso l’analisi genetica, è fondamentale non solo per la comprensione della patogenesi, ma anche per l’individuazione di marcatori da impiegare nelle indagini diagnostiche, prognostiche e come ipotetico bersaglio per terapie farmacologiche. La mia esperienza sperimentale si inserisce in un progetto di ricerca volto alla caratterizzazione mutazionale dei carcinomi poco differenziati, con particolare attenzione ai geni coinvolti nelle vie di trasduzione del segnale implicati nella iniziazione e progressione tumorale di numerosi tumori maligni umani, nonché di quella tiroidea, quali AKt-1 (es. 4), AKt-2 (es. 10, 11), PDK1 (es.10, 14), VEGFR2 (es. 26) e EGFR (es. 18, 19, 20, 21), appartenenti alla via delle MAP chinasi e della PI3K insieme ai geni PDGFRα (es.12, 18) e c-Kit (es. 9, 11, 13, 17). Le tecniche molecolari utilizzate sono: la Real Time PCR-HRMA, la PCR-SSCP e il sequenziamento genomico diretto. L’analisi genica è stata condotta su 20 campioni di DNA estratto da tessuto tumorale congelato, ciascuno dei quali affiancato, quando possibile, all’analisi di DNA estratto da tessuto sano del lobo contro laterale e DNA estratto da metastasi linfonodale. I risultati preliminari non hanno rivelato alcuna variazione di sequenza negli esoni dei geni AKt-1, AKt-2, PDK1, VEGFR2 e negli esoni 18 e 19 del gene EGFR, né nel tessuto tumorale, né nelle corrispettive metastasi, né nel tessuto normale. Sono state invece riscontrate due variazioni di sequenza (germline) degli esoni 20 e 21 del gene EGFR; in dettaglio è stata evidenziata la presenza dello SNP c.2361 G > A (Q787Q; es. 20) in eterozigosi (4/20) e in omozigosi (8/20). Un singolo caso wild-type per la suddetta variazione, è risultato eterozigote per lo SNP c.2508 C > T (R836R; es. 21). L’analisi dei geni PDGFRα e c-Kit è ancora in corso di completamento

A receptor-antibody hybrid hampering MET-driven metastatic spread

Background The receptor encoded by the MET oncogene and its ligand Hepatocyte Growth Factor (HGF) are at the core of the invasive-metastatic behavior. In a number of instances genetic alterations result in ligand-independent onset of malignancy (MET addiction). More frequently, ligand stimulation of wild-type MET contributes to progression toward metastasis (MET expedience). Thus, while MET inhibitors alone are effective in the first case, combination therapy with ligand inhibitors is required in the second condition. Methods In this paper, we generated hybrid molecules gathering HGF and MET inhibitory properties. This has been achieved by 'head-to-tail' or 'tail-to-head' fusion of a single chain Fab derived from the DN30 MET antibody with a recombinant 'ad-hoc' engineered MET extracellular domain (decoyMET), encompassing the HGF binding site but lacking the DN30 epitope. Results The hybrid molecules correctly bind MET and HGF, inhibit HGF-induced MET downstream signaling, and quench HGF-driven biological responses, such as growth, motility and invasion, in cancer cells of different origin. Two metastatic models were generated in mice knocked-in by the human HGF gene: (i) orthotopic transplantation of pancreatic cancer cells; (ii) subcutaneous injection of primary cells derived from a cancer of unknown primary. Treatment with hybrid molecules strongly affects time of onset, number, and size of metastatic lesions. Conclusion These results provide a strategy to treat metastatic dissemination driven by the HGF/MET axis

Low frequency of TERT promoter mutations in a series of well-differentiated follicular-patterned thyroid neoplasms

The diagnostic and clinical approaches to follicular-patterned thyroid neoplasms often create dilemmas for pathologist and clinicians. The molecular analysis of these tumors could be a useful tool to overcome diagnostic limitations. The most frequent molecular alterations are point mutations of RAS family genes. Nevertheless, other molecular markers should be taken into account for their prognostic role, as BRAF mutations and the recently described telomerase reverse transcriptase (TERT) promoter mutation. We investigated the prevalence and the possible role of TERT promoter, BRAF, and RAS mutations in a series of low-risk well-differentiated follicular-patterned thyroid neoplasms. We evaluated 60 follicular adenomas (FA), 29 minimally invasive follicular carcinomas (MIFTC), 82 papillary carcinomas, follicular variant (FVPTC), and 16 noninvasive follicular thyroid neoplasms with papillary-like nuclear features (NIFT-P) for the molecular status of BRAF, H-, N-, K-RAS, and TERT and correlated it with clinic-pathological parameters of tumors. Fiftyseven (30.5%) follicular neoplasms were mutated. In particular, we found 44 RAS mutated neoplasms (23.5%), specifically three FAs, 29 FVPTCs, five NIFT-Ps, and seven FTCs. BRAF mutations were found in ten FVPTCs. Finally, TERT promoter mutations were observed in three FVPTCs and three FTCs; three of them harbored also N-RAS mutations. We confirmed the absence of TERT promoter mutations in benign follicular neoplasms and found a low frequency of TERT promoter mutations in our selected cohort of low-risk follicular-patterned malignancies, speculating their role in the progression and dedifferentiation of thyroid cancer

miRNA expression profiling of ‘noninvasive follicular thyroid neoplasms with papillary-like nuclear features’ compared with adenomas and infiltrative follicular variants of papillary thyroid carcinomas

Follicular variants of papillary thyroid carcinoma include encapsulated (with or without capsular/vascular invasion) and infiltrative forms, which have different clinical behaviors. The encapsulated forms that lack capsular invasion have an indolent clinical behavior that is similar to benign lesions; therefore, they were recently reclassified as ‘noninvasive follicular thyroid neoplasms with papillary-like nuclear features’ (NIFTPs). Because NIFTPs have nuclear features of papillary carcinomas, distinguishing between NIFTPs and infiltrative follicular variant of papillary thyroid carcinoma is almost impossible with cytological examination. The aim of this study is to determine whether miRNA expression profiles may help distinguish between NIFTPs versus follicular adenomas and infiltrative follicular variant of papillary thyroid carcinomas. The expression profiling of 798 miRNAs was tested in 54 thyroid tumors, including 18 follicular adenomas, 19 NIFTPs and 17 infiltrative follicular variant of papillary thyroid carcinomas, using nCounter Nanostring. We found that miR-146-5p, miR-221-5p, miR-222-3p, miR-30e-3p, and miR-152-3p could discriminate between benign and malignant lesions with a very high level of significance (P-value<0.001). High expression levels of miR-146-5p, miR-199a-5p, miR-199b-5p, miR-1285-5p, miR-1915-3p, and miR-4516, and low miR-148b-3p expression were associated with infiltrative growth of follicular variant of papillary thyroid carcinomas. Interestingly, miR-152-3p, miR-185-5p, and miR-574-3p were significantly downregulated in NIFTPs compared with follicular adenomas, whereas miR-10a-5p and miR-320e can discriminate between NIFTPs and infiltrative forms of follicular variant of papillary thyroid carcinomas. In conclusion, a panel of these markers could have high diagnostic potential as well as could be applied to presurgical fine-needle aspiration, especially for lesions classified as indeterminate thyroid nodules.Modern Pathology advance online publication, 2 September 2016; doi:10.1038/modpathol.2016.157

TERT promoter mutations and their correlation with BRAF and RAS mutations in a consecutive cohort of 145 thyroid cancer cases

Papillary thyroid carcinoma (PTC) is the most common type of endocrine malignancy and accounts for ~80% of thyroid carcinomas in adults and 90% in children. Risk stratification is important for identifying patients at higher risk and, for this reason, recent advances in molecular genetics of thyroid cancer can be applied to provide novel biomarkers useful in understanding tumor behavior. B-Raf proto-oncogene, serine/threonine kinase (BRAF) and rat sarcoma (RAS) mutations have been widely studied and appear to have an important role in thyroid tumorigenesis. Somatic telomerase reverse transcriptase (TERT) promoter mutations have been recently identified in several types of malignant tumors, including thyroid neoplasia; however, the actual role of TERT mutations in thyroid tumorigenesis is still under debate. In the present study, the mutational status of BRAF, RAS and TERT was analyzed in order to elucidate the roles of these genes in thyroid tumorigenesis. The TERT mutational analysis was also correlated with an immunohistochemical study of TERT protein expression. According to the literature, our data provide evidence of the BRAF and RAS roles in thyroid tumorigenesis, supporting an association between BRAF (V600E) mutations and the more aggressive clinical and pathological features of thyroid tumors. By contrast, TERT mutations were not significantly associated with any clinical parameters; therefore, its role in initial tumorigenesis should be further investigated

Differences in miRNA expression profiles between wild-type and mutated NIFTPs

Noninvasive encapsulated follicular variants of papillary thyroid carcinomas have been recently reclassified as noninvasive follicular thyroid neoplasms with papillary-like nuclear features (NIFTPs). NIFTPs exhibit a behavior that is very close to that of follicular adenomas but different from the infiltrative and invasive follicular variants of papillary thyroid carcinomas (FVPTCs). The importance of miRNAs to carcinogenesis has been reported in recent years. miRNAs seem to be promising diagnostic and prognostic molecular markers for thyroid cancer, and the combination of miRNA expression and mutational status might improve cytological diagnosis. The aim of the present study was to evaluate the miRNA expression profile in wild-type, RAS- or BRAF-mutated NIFTPs, infiltrative and invasive FVPTCs, and follicular adenomas using the nCounter miRNA Expression assay (NanoString Technologies). To identify the significant Kyoto Encyclopedia of Genes and Genomes (KEGG) molecular pathways associated with deregulated miRNAs, we used the union of pathways option in DNA Intelligent Analysis (DIANA) miRPath software. We have shown that the miRNA expression profiles of wild-type and mutated NIFTPs could be different. The expression profile of wild-type NIFTPs seems comparable to that of follicular adenomas, whereas mutated NIFTPs have an expression profile similar to that of infiltrative and invasive FVPTCs. The upregulation of 4 miRNAs (miR-221-5p, miR-221-3p, miR-222-3p, miR-146b-5p) and the downregulation of 8 miRNAs (miR-181a-3p, miR-28-5p, miR-363-3p, miR-342-3p, miR-1285-5p, miR-152-3p, miR-25-3p, miR-30e-3) in mutated NIFTPs compared to wild-type ones suggest a potential invasive-like phenotype by deregulating the specific pathways involved in cell adhesion and cell migration (Hippo signaling pathway, ECM-receptor interaction, adherens junction, regulation of actin cytoskeleton, fatty acid biosynthesis and metabolism)